Spring 2010 Eukaryotic Cell and Developmental … Dossier...MG 602, Spring 2010 Lab Manual page 3 MG 602 Spring 2010 Eukaryotic Cell and Developmental Biology Laboratory Lab Manual

MG 602, Spring 2010 Lab Manual page 1

MG 602 Spring 2010

Eukaryotic Cell and Developmental Biology

Laboratory

Lab Manual


Table of Contents Staff and Contact Information:…………………………………….………. 3 Introduction:………………………………………………………..………. 4 Lab Notebook and Report Guidelines:…………………………………….. 5 Course Outline:…………………………………………………………….. 6 Background:………………………………………………………………... 8 Overview of Experiments:…………………………………………………. 16

Analysis of Cell Membranes and the Cytoskeleton………………... 17 Microtubules and Cellular Organization…………………………… 18 Centrosomes and Centriole-associated Structures………………… 19 Construction of a DsRed-tagged Expression Construct…………… 20

Generation of siRNA Pools……………………………………….. 21 Sterile Technique and Guidelines for Cell Culture………………… 22 Instructions for Passaging Cells…………………………………… 23

HeLa-GFPCetn2 Cells and Tet-regulated Expression……………... 24 Laboratory Exercises:……………………………………………………... 25 Tuesday, 3/30..……………………………………………………... 26 Thursday, 4/1……………………………………………………... 27 Tuesday, 4/6………………………………………………………... 28 Thursday, 4/8………………………………………………………. 30 Tuesday, 4/13………………………………………………………. 32 Thursday, 4/15……………………………………………………... 34 Tuesday, 4/20………………………………………………………. 35 Thursday, 4/22……………………………………………………... 37 Tuesday, 4/27………………………………………………………. 38 Thursday, 4/29……………………………………………………... 39 Tuesday, 5/4………………………………………………………. 41 Thursday, 5/6……………………………………………………... 44 Monday, 5/10………………………………………………………. 45 Tuesday, 5/11………………………………………………………. 47 Thursday, 5/13……………………………………………………... 48 Monday, 5/17………………………………………………………. 50 Tuesday, 5/18………………………………………………………. 51 Thursday, 5/20……………………………………………………... 53 Tuesday, 5/25………………………………………………………. 56 Thursday, 5/27……………………………………………………... 58 Protocols:…………………………………………………………………... 60 Protocol #1: Fixing Cells on Coverslips ………………………….. 60 Protocol #2: Mounting coverslips ………………………………… 61 Protocol #3: Indirect Immunofluorescence (IIF) Analysis ………... 62 Protocol #4: Effectine Transfection of Plasmid DNA ……………. 63 Protocol #5: Bacterial Transformation ……………………………..64 Protocol #6: Plasmid “Miniprep” DNA Isolation ………………… 65 Protocol #7: Coating Coverslips With Polylysine ………………… 66


MG 602 Spring 2010

Eukaryotic Cell and Developmental Biology Laboratory

Lab Manual

Instructor: Harold A. Fisk Office: BSB 272 (office hours by appointment) Phone: 292-0318 email: [email protected] TA’s: Carla Justiniano Office: BSB 216 (office hours by appointment) Phone: 247-8715 email: [email protected] Amanda Bliemeister Office: BSB 216 Office hours: Phone: 247-8715 email: [email protected]

Sheng-Wei (Sara) Chang Office: BSB 216 Office hours: Phone: 247-8715 email: [email protected] Ching-Hui (Julia) Yang Office: BSB 216 Office hours: Phone: 247-8715 email: yang.1030@ buckeyemail.osu.edu

Christopher Kasbek Office: BSB 216 Office hours: Phone: 247-8715 email: kasbek.1@ buckeyemail.osu.edu Coordinator: Jeff Bils Office: BSB 215 Phone: 688-4227


Introduction Welcome to MG 602, a laboratory course that will expose you to a variety of molecular genetic techniques. At the heart of this course is the application of modern molecular genetics approaches to cultured human cells. However, you will also grow bacterial cultures as you learn additional approaches in modern molecular biology in order to prepare some of the reagents you will use for the experiments in the course. The culture of human cells requires medium that is very complex and rich in nutrients, and is very easy to contaminate with bacteria. Under ideal conditions bacteria divide every 30 minutes, while it takes human cells between 18 and 48 hours to divide once. In that time a single bacterial cell can divide 48-96 times yielding between 68 and 136 trillion (6.8x1010-1.3x1011) cells.

It is therefore VERY important to keep cultures of bacteria and human cells separate. You will be multitasking to a significant degree during this course, and there will be days when you must culture both bacteria and human cells. Accordingly you must keep these different types of cultures. In order to avoid contaminating your human cells with bacteria, adhere to the following guidelines: 1) NEVER HAVE BACTERIAL AND HUMAN CELL CULTURES OUT AT THE SAME TIME, 2) Practice sterile technique (described below) at all times. 3) Decontaminate surfaces after using bacteria AND before using tissue culture. 4) When possible, perform any bacterial operations at the end of the day AFTER any tissue culture. Grading

This is a techniques based laboratory course, and your grade will reflect your participation. However, you will not be graded strictly on the outcome of your experiments. We will be addressing original research problems, the answers for which are not known, and you often will not know what the outcome of your experiment is “supposed” to be. However, you must demonstrate that you understand each experiment and be able to discuss reasons why your results might be different than predicted. Accordingly, 60% of your grade will be determined by lab participation, and 40% of your grade will be determined by two exams. Your laboratory performance grade will consist of your laboratory notebook (graded twice), your average on eight weekly quizzes (normalized to 10 points), and two lab reports. Attendance is mandatory and absences will only be permitted with a valid excuse. No makeup will be given for the quizzes or exams under any circumstances, and unexcused absences will not be tolerated.

Grading: 40% Exams 60% Lab participation Midterm Exam, week 5 40 pts Lab Notebook week 5: 20 pts Final Exam, week 10 40 pts Lab Notebook week 10: 20 pts

Lab Report#1: 20 pts Lab Report#2: 20 pts Quizzes: 10 pts Homework: 30 pts

Academic Integrity The Ohio State University and the Committee on Academic Misconduct expect that all students have read and understand the University’s Code of Student Conduct. Students must recognize that failure to follow the rules and guidelines established in the University’s Code of Student Conduct may constitute Academic Misconduct. If you have any questions about this policy or what constitutes academic misconduct in this course, please contact the instructors. Of particular concern for this course is the lab report; while you can discuss the preparation of the report with your lab partner, your lab report must be exclusively your own work. For more information on academic misconduct please see the Committee on Academic Misconduct web pages (oaa.osu.edu/coam/home.html).


Lab Notebook Guidelines Your lab notebook must be well organized, although we won’t be enforcing a particular format. You must make daily entries for each experiment. We will be working on multiple experiments for most days, and for each day you must make entries for each of the experiments we work on for that day. The first time we work on an experiment, there should be a description of the overall purpose and goals of the entire experiment, followed by specific entries for exactly what was done on that day. On subsequent days that experiment is worked on there need be only specific entries for what was done on that day. Regardless of exactly how you choose to arrange your notebook entries, it will require good organizational skills because we will be working on multiple experiments most days. Overall your notebook should consist of the following sections:

1) Table of contents noting the location of: i. Overall description for each major experiment ii. Each daily entry iii. Overall conclusion for each major experiment (an example Table of Contents will be posted on Carmen) 2) Overall description of each major experiment including

i. Title ii. Purpose iii. Materials and Methods

3) Daily entries for each experiment including i. Experiment name ii. Purpose of that day’s experimentation iii. Materials and Methods for that day iv. Observations v. Results vi. Conclusions

4) Conclusions to each major experiment i. Title ii. Results iii. Conclusions

Lab Report Guidelines You will also be required to submit two lab reports, one for each five-week portion of the course. It should include the same general sections as your lab notebook, but must combine all three experiments into a single integrated report. For example, you must come up with a single statement of “Purpose” that encompasses all three experiments. Note that while you may consult with your lab partner as you prepare your report, the written report must be your own work. In past years lab partners have come dangerously close to plagiarism, and this will not be tolerated. Lab Report Organization

i. Title ii. Purpose iii. Materials and Methods iv. Results v. Conclusions


Course Outline: Week 1: Introduction to Cellular Organization (Exp 1) Tu 3/30 Stain cells with WGA, Paclitaxel, and Phalloidin (Exp 1) Th 4/1 Microscope Tutorials Examine stained cells to visualize membranes and the cytoskeleton Homework, design PCR primers for TOPO cloning (Exp 5) Week 2: Indirect Immunofluorescence (IIF) and Transfection (Exp 2) Tu 4/6 Fix cells and perform IIF (Exp 2a) Transfect cells with fluorescently tagged Tubulin (Exp 2b) Turn in Homework (Exp 5) email Primer Sequences to TAs before start of class Th 4/8 Group A: Group B: 1) Examine IIF (Exp 2a) 1) Set up PCR reactions (Exp 5) 2) Set up PCR reactions (Exp 5) Fix Transfected cells (Exp 2b) Fix Transfected cells (Exp 2b) Perform IIF Perform IIF Set up D-TOPO cloning (Exp 5) Set up D-TOPO cloning (Exp 5) 2) Examine IIF (Exp 2a) Week 3: Centrosomes and Cellular Organization (Exp 3) Tu 4/13 Treat Cells with Nocodazole (Exp 3) Fix and perform IIF Transform D-TOPO cloning reactions (Exp 5) Th 4/15 Group A: Group B: 1) Pick minipreps (Exp 5) 1) Examine IIF (Exp 2a, 3) T7 PCR primer homework (Exp 6) 2) Pick minipreps (Exp 5)

2) Examine IIF (Exp 2a, 3) T7 PCR primer homework (Exp 6)

Week 4: Centrosome Associated Structures (Exp 4) Tu 4/20 Transfect GFP-Cetn2 cells with RFP-tagged centrosome proteins (Exp 4) Isolate plasmid DNA, set up restriction digests (Exp 5) Th 4/22 Fix and stain transfected cells (Exp 4) Gateway® Recombination reaction and bacterial transformation (Exp 5) Homework Finding homologues of centrosomal proteins Week 5: Completion of Expression Constructs (Exp 5), siRNA Production (Exp 6) Tu 4/27 Set up first PCR for siRNA (Exp 6)

Group A: Group B: 1) Examine transfections (Exp 4) 1) pick minipreps (Exp 5)

2) pick minipreps (Exp 5) 2) Examine transfections (Exp 4)

Th 4/29 Midterm Exam! Set up in vitro transcription of PCR products for siRNA production (Exp 6)

Isolate plasmid DNA, set up restriction digests (Exp 5)


Week 6: Cell Counting and Preparation for Transfection Tu 5/4 Learn cell culture (Exp 5, 6) Purify dsRNA from transcription reactions (Exp 6) Cleave dsRNA into siRNA using RNAseIII (Exp 6) Purify siRNA (Exp 6) Th 5/6 Passage cells for Monday (Exp 5)

Count cells and plate at specific cell densities Coat coverslips (Exp 5) Week 7: Localization of Candidate Centrosome Proteins (Exp 5) Mo 5/10 Come in after noon to passage HeLa cells for Tuesday (Exp 5) Tu 5/11 Transfect cells with GFP expression construct (Exp 5a) Passage cells Fix and stain cells for colony counting Th 5/13 Passage cells for Monday (Exp 6)

Harvest transfections (Exp 5a) Perform IIF (Exp 5a)

Coat coverslips (Exp 5) Week 8: S-phase Arrest (Exp 5) and Depletion of Candidate Centrosome Proteins (Exp 6) Mo 5/17 Come in after noon to passage HeLa cells for Tuesday Tu 5/18 Transfect cells with siRNA (Exp 6) Transfect cells with expression construct (Exp 5b) Arrest plasmid transfected cells in S-phase (Exp 5b) Th 5/20 Harvest cells for IIF and western (Exp 5b, 6) Week 9: Depletion of Candidate Centrosome Proteins, Continued (Exp 6) Tu 5/25 Run SDS-Page gels of siRNA samples (Exp 6)

Transfer gels to nitrocellulose (Exp 6) Th 5/27 Process western blots (Exp 6) Week 10: Collect Final Data, Second Exam Tu 6/1 Scan western blots

finish looking at IIF (any remaining experiments)

Th 6/3 Second Exam


Background Cell Theory holds that the cell is the basic unit of life. Indeed, the simplest forms of life capable

of self-replication are single celled organisms. The only simpler life forms are viruses that cannot replicate outside of a host cell, and more complex organisms are nothing more than collections of specialized cells. While a complex organism such as a human depends on the function of organs and tissues, these organs and tissues are simply collections of specialized cells. Accordingly, the functions of an organ or tissue depend on the function of its constituent cells, and all defects in organ or tissue function are caused by some defect at the cellular level. Anyone who hopes to understand human health must therefore have a fundamental understanding of cell biology. The History of Cell Biology

Robert Hooke was the first person to recognize cells as individual compartments when he examined sections of cork tissue under his microscope in 1665, and Antoine van Leeuwenhoek was the first person to observe a living cell when he made observations on single celled algae in 1674. More than a century later in 1811 Ludolph Christian Treviranus proposed that plants were made up of individual units separated by an intercellular space, and Johann Jacob Paul Moldenhawer demonstrated the validity of this assertion in 1812 by developing methods for separating individual cells. Based on these and his own observations, Henri Dutrochet proposed in 1824 that “The cell is the fundamental element of organization,” although credit for modern Cell Theory is typically given to Theodor Schwann, Matthias Schleiden, and Rudolf Virchow. Schleiden and Schwann proposed Cell Theory in 1839, but incorrectly theorized that cells arose through “Free Cell Formation,” and in 1858 Virchow demonstrated that cells form by division of existing cells. The three tenets of modern Cell Theory are; 1) All living things are composed of cells; 2) Cells are the basic unit of structure and function within organisms; and 3) Cells arise by division of existing cells. Simply put, Cell Theory states that cells are the basic unit of life. The discipline of Cell Biology grew out of Cell Theory and is the study of cells and their function, while the closely related field of Developmental Biology is the study of the development and differentiation of cells into the specialized forms that give organs and tissues their structure and function. Cellular Organization

While Cell Theory recognizes that cells are the smallest compartments capable of self-replication, cells are themselves highly compartmentalized. The internal contents of a cell (or cytoplasm, cyto- meaning cell and -plasm meaning substance) are separated from the external environment and partitioned into distinct compartments by a series of biological membranes formed by lipid bilayers (Figure 1). The hydrophobicity of a lipid bilayer creates a barrier to the movement of water-soluble material, just as a balloon provides a barrier to the movement of gasses. The plasma membrane separates the cytoplasm from the extra cellular milieu, allowing the chemical composition inside the cell to be distinct from that on the outside. Cytoplasm contains a number of large structures that give each cell type its unique shape and function and are suspended in a fluid cytosol. Cytosol consists of water, soluble proteins, salts, minerals, and small organic molecules. The remainder of the cytoplasm consists of a variety of large protein complexes and a number of organelles (“little organs”) that perform basic metabolic functions. The protein complexes in the cytoplasm include the cytoskeleton that provides cellular organization, and machines such as ribosomes and proteasomes responsible for the synthesis and degradation of proteins, respectively.

The organelles present in vertebrate cells include the endoplasmic reticulum, the Golgi complex, endosomes, lysozomes, peroxisomes, mitochondria, and the nucleus, and each organelle performs a specific subset of metabolic functions. Proteins destined for specific organelles, the plasma membrane, or for secretion from the cell are inserted into the endoplasmic reticulum as they are synthesized, and transported from there to the Golgi or the nuclear envelope, which is contiguous with the endoplasmic reticulum. The Golgi modifies proteins with oligosaccharides and sorts them to peroxisomes or


endosomes. Peroxisomes contain enzymes called peroxidases that detoxify harmful peroxides and metabolize fatty acids. Endosomes sort proteins to the final destinations of the plasma membrane, extracellular space, or lysozomes. Endosomes are also the site where material internalized from the plasma membrane or extra cellular space is sorted to lysosomes. Lysozomes contain digestive enzymes that recycle cellular components, digest nutrients from the extra cellular environment, and destroy bacteria, viruses, and other foreign materials. Mitochondria are the powerhouses of the cell where energy needed for cellular processes is produced. The nucleus is the information center of the cell, where the cell’s genetic information is transcribed into RNA, which is then transported into the cytoplasm to participate in the production of cellular proteins.

Subcellular organelles create several distinct chemical environments within the cell, because their internal environments are physically separated from the cytoplasm by a lipid bilayer. For example, the interior of lysozomes and endosomes is more acidic than the cytoplasm, and the low pH of these organelles is essential for the metabolic processes that take place there. While all organelles have a membrane, three organelles have a membrane structure that is more complex than that of the plasma membrane. Lysozomes have a complex membrane structure that is the result of their ability to internalize other organelles. In contrast, mitochondria and nuclei have a double membrane and also contain their own genomes, suggesting that they arose through endosymbiosis (the process of one organism evolving to live inside a second organism). The Cytoskeleton

Organelles are typically found at specific locations within the cell. This, along with overall organization and architecture, is maintained by the cytoskeleton. The cytoskeleton is composed of three different types of filamentous networks, each formed by a different type of protein that can self-associate to form long filaments with different properties. These filaments were initially defined by electron microscopy and named according to their size. The smallest of the three are helical filaments of roughly 7

Figure 1. Organization of a typical vertebrate cell. Adapted from Molecular Biology of the Cell, 4th Ed., Fig 1-31


Figure 2. Centrosomes and Centrosomal Proteins.

nm diameter called microfilaments, while intermediate filaments are roughly 10 nm in diameter, and microtubules are hollow cylinders roughly 25 nm in diameter.

Each of these filamentous systems is composed of a distinct subset of proteins. Microfilaments are composed of actin, which can polymerize through both lateral and head-to-tail associations to form a helical filament two actin molecules in diameter. Once polymerized into filaments, actin fibers can form two different types of networks - parallel bundles or antiparallel meshworks. The basic unit of microtubule structure is a heterodimer consisting of two related proteins, α- and β-Tubulin. The α/β heterodimer can polymerize in a head-to-tail fashion to form an unstable protofilament, and lateral associations between protofilaments leads to the assembly of a hollow cylinder. In a test tube the number of protofilaments in a microtubule is variable, but inside living cells microtubules invariably have thirteen protofilaments. Intermediate filaments are heterogeneous, and can be formed by many types of proteins such as keratins, vimentin, neurofilaments and lamins. The basic structural unit of intermediate filaments is an anti-parallel dimer (either a homodimer or a heterodimer). These dimers then form both lateral and head-to-tail associations to form filaments of variable length and diameter.

The assembly of microfilaments and microtubules is energy-dependent, requiring ATP or GTP, respectively, and both filaments are polarized with a distinct head and tail. In contrast, intermediate filament formation does not require energy, and results in inherently non-polar structures. The distribution of the three networks is highly interdependent. For example, intermediate filaments are transported toward the cell periphery along microtubules, and toward the cell center along microfilaments. Centrosomes:

Microtubules are unique among the three cytoskeletal networks in that they are highly dynamic and are largely (though not uniquely) formed at a single location within the cell called the Microtubule Organizing Center or MTOC. In vertebrate cells the primary MTOC is a structure called the centrosome. The centrosome consists of a pair of centrioles, cylindrical arrays of triplet microtubules with nine-fold symmetry, surrounded by a pericentriolar matrix (PCM) responsible for the ability of the centrosome to act as an MTOC. Centrosomes are present at just one or two copies per cell (depending on the stage of the cell cycle). They were discovered in 1888 by Theodore Boveri, who named them for their association with a structure found at the center of the cell called the “aster,” one of the most prominent cellular features detectable with early microscopes. The structure of centrioles is analogous to that of basal bodies that template cilia and flagella, and centrioles and basal bodies are interconvertable. Upon fertilization of the oocyte, the sperm basal body is converted into a centriole, which then recruits PCM to become a centrosome. Once a cell has terminally differentiated, the oldest of the two centrioles, called the mother centriole, is converted back into a basal body so that it can template the assembly of the primary cilia. In specialized cells such as spermatocytes, the basal body templates the assembly of a flagella, completing the circle. As we have learned more about the function of centrosomes,


Figure 3. The Mitotic Spindle

Figure 4. Multipolar Mitosis

it has become apparent that the name given by Boveri was quite appropriate; centrosomes participate in many cellular functions and are of central importance to cell division. The assembly of centrosomes is at the center of MG602, where we will study the centrosome as we learn to use modern molecular genetic tools used in cell biology. The distribution of several centrosomal proteins (some of which we will be looking at in MG602) with respect to centrioles and the pericentriolar matrix is summarized in Figure 2.

The association of centrosomes with asters explains why they are best known as “Microtubule-organizing centers;” we know today that centrosomes are required for aster formation, and that asters are composed of microtubules. The PCM houses many proteins that regulate microtubule dynamics, and it is the PCM that is responsible for the microtubule-organizing capacity of centrosomes. Microtubules are made entirely of two proteins, α-Tubulin and β-Tubulin, which together form a heterodimer that is the basic unit of microtubule structure. The rate-limiting step in microtubule assembly consists of the head-to-tail association of α/β heterodimers into protofilaments, and the lateral association of protofilaments into a tubule, collectively termed nucleation. In a test tube microtubule nucleation cannot occur if the amount of Tubulin is below a threshold level, called the critical concentration. The concentration of Tubulin inside cells is well below the critical concentration, but The PCM contains a third Tubulin family

member called γ-Tubulin (discovered here at OSU in Dr. Berl Oakley’s lab) that is not incorporated into microtubules, but instead forms a ring-shaped complex that provides a template for nucleation. Therefore, centrosomes effectively lower the critical concentration, allowing microtubule nucleation to occur in a highly regulated manner at very low levels of Tubulin. Accordingly, centrosomes organize the microtubule cytoskeleton that is important for many aspects of cell shape, motility, and growth. Centrosomes also coordinate the re-organization of microtubules into the mitotic spindle apparatus in preparation for cell division (Figure 3).

Centrosomes also have additional functions not strictly related to their identity as microtubule organizing centers. Centrosomes are required for the G1/S transition and cytokinesis, and thus regulate the decisions to start and complete the cell cycle. These later functions are

dependent on centrioles, as opposed to the PCM. Through cytokinesis cells normally inherit a single centrosome. This single centrosome must be precisely duplicated before entering mitosis in order to form the mitotic spindle. If too many centrosomes are produced during the duplication process, the extra centrosomes will cause the formation of aberrant mitotic spindles with more than two poles (Figure 4). These multipolar spindles cannot segregate chromosomes properly and generate daughter cells that do not have the correct number of chromosomes. The state of having the incorrect complement of chromosomes is called aneuploidy, and is observed in the majority of human tumors. In fact, many human tumors also have extra centrosomes that are thought to be important in tumorigenesis. Because the production of multipolar spindles can have such drastic consequences, it is critical to understand the mechanisms that control centrosome duplication. Two cellular processes that are known to be important for centrosome duplication in human cells are protein phosphorylation and proteasome-mediated degradation. Protein Phosphorylation:

Proteins are produced by transcription of genomic DNA into a messenger RNA molecule (mRNA), followed by translation of this mRNA into protein. After translation, the resulting proteins are often altered by a variety of enzymes. Called post-translational modification, such alterations can change


Figure 6. The Ubiquitin-Proteasome Pathway.

Figure 5.

the structure, function, or localization of proteins. Perhaps the most common and versatile of these post-translational modifications is phosphorylation. Phosphorylation is catalyzed by enzymes called protein kinases that transfer the gamma phosphate moiety from adenosine triphosphate (ATP) to the hydroxyl group of serine, threonine, or tyrosine residues in protein substrates (see Figure 5). One reason protein phosphorylation is so versatile is that it is reversible; the phosphate moiety can be removed by enzymes called protein phosphatases. The phosphate moiety (PO4

2-) is electron dense and negatively charged, and its addition to a protein can influence the protein’s structure and/or function. This structural change can alter either the activity of the phosphorylated protein, or change the way the phosphorylated protein interacts with other proteins. For example, phosphorylation is responsible for turning the catalytic activity of many protein kinases both on and off. However, phosphorylation can also change the way a protein interacts with other proteins. For example, sequential phosphorylation of the yeast centrosomal protein Spc42 by multiple protein kinases allows it to interact with other centrosomal proteins and to assemble into the centrosome structure. Several protein kinases are found at centrosomes in human cells, and phosphorylation is very important in the centrosome duplication process. One of these centrosomal protein kinases is the Mps1 protein kinase, and Mps1 exemplifies the importance of phosphorylation in centrosome duplication. Mps1 phosphorylates several centrosomal proteins, and the catalytic activity of Mps1 is required for centrosome duplication. Although the precise mechanisms are not well worked out phosphorylation of proteins by Mps1 presumably allows them to be assembled into centrosomes. However, as discussed below Mps1 is itself also regulated by phosphorylation. Proteasome-mediated Degradation:

The proteasome is a large cellular complex containing many proteases, and is the site of regulated protein destruction. The proteasome can be found in many cellular locations, including the centrosome, and there are many requirements for proteasome activity in centrosome duplication. The trigger for proteasome-mediated degradation is typically the covalent modification of target proteins with the small molecule ubiquitin (see Figure 6). Ubiquitin is transferred to target proteins through an enzymatic cascade. Ubiquitin is activated by an E1 (Ubiquitin Activating) enzyme, transferred to an E2 (Ubiquitin Conjugating) enzyme, then to an E3 (Ubiquitin Ligase) enzyme, and finally to the substrate protein. The E1 enzyme requires energy to activate ubiquitin, but the subsequent cascade is energy independent. E1’s, E2’s, and E3’s are often found together in large complexes, and substrate specificity in this cascade is a function of the E3’s. There are several classes of E3s. One class is composed of many different proteins, and is exemplified by the SCF (named for the Skp, Cullin, and F-box proteins of which it is composed) and the Anaphase Promoting Complex/ Cyclosome (APC/C). Other E3’s have been described as


soluble monomeric enzymes, such as c-Cbl that ubiquitinates the Epidermal Growth Factor Receptor and ZAP70 tyrosine kinases. However, there is mounting evidence that all E3’s interact with cofactors, and c-Cbl forms a complex with at least one other protein called STS-1 that modulates the activity of c-Cbl. While ubiquitination itself is reversible, proteasome-mediated degradation is a non-reversible event and as such often serves as a trigger for major events. As examples, the proteasome-mediated degradation of a Cyclin-dependent kinase inhibitor by the SCF drives cells into S-phase, and degradation of an anaphase inhibitor by the APC/C drives cells into anaphase. However, proteasome-mediated degradation can also provide tight control over the levels of regulatory proteins, as is the case for c-myc and Mps1. For example, an SCF E3 that contains the Fbw7 F-box protein targets c-myc for proteasome-mediated degradation. The protein kinase ERK phosphorylates c-myc at threonine residue 62 (T62), and this phosphorylation transiently suppresses the degradation of c-myc. Because phosphorylation is reversible c-myc degradation is only suppressed transiently. Similarly, phosphorylation of Mps1 by Cdk2 suppresses the proteasome-mediated degradation of Mps1. However, the E3 ligase required for Mps1 degradation has not been identified. Limitations of Human Cells:

Molecular genetic techniques have provided very powerful tools for biologists working in model organisms (i.e. budding yeast, fruit flies, and worms), and have contributed greatly to our understanding of a variety of cell biological and developmental problems. These molecular genetic approaches include the ability to isolate mutations in virtually any gene, the ability to clone genes, the ability to re-introduce cloned genes into the host organism, and the ability to manipulate the host genome at the molecular level. This latter ability is particularly powerful because it allows the researcher to quickly delete and/or replace any gene of interest. However, the application of these types of molecular genetic approaches to human cells has been lacking. Clearly, it is not possible to create mutations in humans, and informative diseases for which the genetic basis is known are very rare. While there are no ethical issues with making mutations, deleting, or replacing genes in cultured human cells, it is impractical, time consuming, and/or costly to do so. Procedures for deleting and replacing genes are well developed in mice, but again it is a cumbersome, time consuming, and costly process. For this reason, many advances in our understanding of human biology have actually come from studies performed in model organisms. For example the fruit fly Drosophila melanogaster is a very powerful genetic system that is used in MG601. Because mutations that disrupt the function of almost any gene can be identified, and because the mutant genes and genomes can be easily manipulated, model organisms have been used to great effect to identify proteins involved in cellular processes such as centrosome duplication that are important in human diseases such as cancer. Accordingly, budding yeast and fruit flies have greatly aided our overall understanding of centrosome duplication, while advances on this problem in human cells themselves have been slow. However, the recent advent of proteomics and RNA interference techniques has opened mammalian systems to molecular genetic approaches. Proteomics:

Proteomics is the use of mass spectroscopy to identity proteins, and can rapidly determine which proteins are present in complex mixtures. Mass spectroscopic techniques have been around for nearly a century, but have only recently been applied to biological molecules. Whole proteins are large and beyond the mass that can be accurately measured using a mass spectrometer. However, proteases such as trypsin, which cleaves proteins at any lysine or arginine residue, can be used to cut proteins into smaller peptides fragments. Trypsin cuts proteins reproducibly to generate peptides with a characteristic mass that can be accurately measured using a mass spectrometer. Two recent advances have allowed mass spectroscopic techniques to be applied to the identification of biological molecules. First, many genomes, including our own, have now been completed sequenced. Second, advances in computing power have allowed the generation of algorithms that can predict the mass of every possible tryptic peptide from the sequence of any genome. Remarkably, the mass of tryptic peptides is nearly unique, and very few have


Figure 7. Mps1 Localizes to Centrosomes. MCF7 cells expressing GFP-Cetn2 (green) stained with an antibody against Mps1 (red).

the same mass. Therefore, the presence of as few as three peaks on a mass spectrometer that match the masses of predicted tryptic peptides from a human gene is sufficient to conclude that the protein encoded by that gene is present in the mixture placed in the mass spectrometer. Accordingly, mass spectroscopy is a powerful method for the identification of proteins from complex mixtures. As an example, a recent study subjected purified mammalian centrosomes to a proteomic analysis and identified roughly 500 proteins, most of which had never previously been documented to be at centrosomes. However, many of these proteins are contaminants that do not represent bona fide centrosome proteins, making it necessary to examine the centrosomal localization and function of each protein in this collection. While this may seem daunting, it is in fact a historical advance that has identified a list of 500 candidate centrosomal proteins from among the 20,000-40,000 proteins estimated to be encoded by the human genome. RNA interference:

RNA interference is a technology for gene silencing that was accidentally discovered by researchers using antisense RNA molecules to disrupt gene function in worms. Antisense RNAs can be used to deplete a protein of interest because they base-pair with the mRNA encoding that protein and prevent its translation. In contrast sense RNAs cannot base pair with the mRNA, and therefore provide an appropriate negative control for antisense RNA experiments. Short, chemically synthesized RNA molecules were typically used because long RNAs are cytotoxic to vertebrate cells. In contrast, it was found that long RNAs were not toxic to worms, allowing researchers to use longer RNA molecules that were more effective and could be easily made in the laboratory by in vitro transcription. However, sense RNA molecules made by in vitro transcription were often as potent as the antisense molecules themselves. It was soon found that the active molecule in these preparations was actually double stranded RNA (dsRNA) formed by hybridization of the template with the complementary strand synthesized in vitro. This lead to the discovery of the RNA induced Silencing Complex (RISC). The RISC is a sequence-specific RNA degradation machine that binds to any dsRNA present in the cell and degrades any mRNAs with complementarity to the dsRNA. In nature, dsRNA is typically found in viral genomes, and the RISC is thought to reflect an evolutionarily conserved anti-viral response that destroys viral mRNAs. The pioneering work in worms quickly led to the use of small interfering RNA molecules (siRNAs) in human cells. These siRNAs are 21-25 base pair dsRNA molecules that can be introduced into human cells. By designing siRNAs that are complementary to a gene of interest researchers can easily deplete the mRNA encoded by that gene. Because no new protein can be made without the mRNA, RNA interference is a quick and easy way to deplete a protein from a cell without the need to generate a gene deletion.

MG 602; The Centrosome as a Model for Molecular Genetic Studies in Human Cells:

True genetic experiments are nearly impossible in human cells, and this has historically made it difficult to study many processes in human cells. However, proteomics and RNA interference can be used to quickly identify novel proteins and determine their function. In the first half of MG602, we will use the mammalian centrosome as an example of how a basic cell biological problem can be dissected in mammalian cells. The exercises in this first part of the course will examine the centrosomal localization and functions of four candidate centrosome proteins that were identified in the proteomic analysis of the


Figure 8. STS-1, a new regulator of Mps1 degradation. (A) HeLa cells expressing GFP-tagged STS-1 (green) were stained with an antibody to γ-Tubulin (red). (B) The siRNA-mediated depletion of STS-1 leads to increased Mps1 levels.

A.

B. Con. STS-1

centrosome. The specific proteins were chosen for their relevance to centrosome duplication, protein phosphorylation, and proteasome-mediated degradation.

The Mps1 protein kinase localizes to centrosomes, specifically to the centrioles themselves (see Figures 2 and 7). Mps1 is required for centrosome duplication, and centrosome duplication is very sensitive to the amount of Mps1 at centrosomes. Mps1 levels are regulated by proteasome-mediated degradation that occurs specifically at centrosomes. The cell cycle regulator Cdk2 phosphorylates Mps1, which suppresses the degradation of Mps1 allowing its accumulation at centrosomes for a brief period during which it drives centrosome duplication. Normally, Mps1 only accumulates at centrosomes transiently, because Cdk2 is transient and Mps1 phosphorylation is reversible. However, the failure to reverse Cdk2 phosphorylation prevents the degradation of Mps1 and causes the production of extra centrosomes (cover photo panel C). We have found that PP2A, confirmation-sensitive, proline-directed protein phosphatase, reverses the Cdk2-dependent phosphorylation of Mps1, but the molecules that target Mps1 for degradation are not known. Previous students in MG602 found that the proteins STS-1 and ZAP-70 are bona fide centrosomal proteins that are required for Mps1 degradation (Figure 8). STS-1 and ZAP-70 were identified in the proteomic analysis of the centrosome, but nothing was known about their function at centrosomes. Therefore, students in MG602 have contributed to major discoveries that have elucidated the regulation of centrosome duplication.

We will perform a similar analysis this year, examining five candidate centrosome proteins. Two of these proteins, VDAC1 and VDAC2, were chosen because they are related to VDAC3. The three remaining proteins, CLIC1, FAM161a, and FKBP3, were chosen because they were identified in the proteomic analysis of the centrosome and are potentially relevant to the regulation of Mps1. Although VDAC3 is an anion channel found in the mitochondrial outer membrane, we isolated it as a protein that binds to Mps1, and have also shown that it is present at centrosomes where it is required for the accumulation of Mps1 at centrosomes. VDAC1 and VDAC2 are highly related to VDAC3, and we will test whether they are also present at centrosomes and interact with Mps1. CLIC1 is also an anion channel, and we will test the hypothesis that it has a centrosomal function similar to that of VDAC3. FKBP3 is a prolyl isomerase, and we will test the possibility that FKBP3 regulates centrosome duplication by rendering Mps1 sensitive to PP2A. FAM161a is a protein of unknown structure and function, and we will attempt to determine if this protein has any centrosomal function.

For the experiments in this course, each group will take a cDNA encoding one of these five proteins, CLIC1, FAM161a, FKBP3, VDAC1, or VDAC2. You will use this cDNA for recombination based cloning to generate a fusion with the red fluorescent protein from the pink mushroom coral Discosoma sp. (DsRed), and as a template to generate gene-specific siRNAs. You will then express the DsRed-tagged protein in human cells to determine if it localizes to centrosomes, and use the siRNAs in RNA interference experiments to determine if your candidate protein has any function in centrosome assembly and/or Mps1 degradation (as shown for STS-1 in Figure 8).


Overview of Laboratory Exercises and Experiments You will be performing six major experiments over the course of the quarter. Because of the complex time lines these experiments will overlap, and you will often find yourselves working on more than one experiment on any given day. You will be given cells for the first four experiments so that you can learn the techniques you will need for the final two experiments, for which you will generate the critical reagents and propagate the cells yourselves. To accomplish this you will have to come in outside of normal class hours on several occasions to maintain cultures and passage cells for experiments. We have organized the course so as to minimize this outside work, and to best accommodate your varied schedules you will work in groups of two. Below are brief descriptions of each experiment, followed by the course outline and specific protocols for each experiment. Experiment 1: Fluorescent analysis of cell membranes and the cytoskeleton In the first week, you will stain cells with phalloidin to reveal the microfilament network, and with wheat germ agglutinin (WGA) to visualize both internal and external cell membranes. Experiment 2: Analysis of cellular structures by Indirect Immunofluorescence In the second week, you will use antibodies against specific cellular proteins to examine the distribution of a variety of cytoplasmic structures. Experiment 3: Microtubules and cellular organization In the third week, you will use IIF to study the role of microtubules in cellular organization by examining cells treated with a microtubule poison called Nocodazole. Experiment 4: Centrosomes and associated structures In the fourth week, you will use IIF to examine the distribution of a variety of proteins within centrosomes, including those found at centrioles, centriolar appendages, and the pericentriolar material. Experiment 5: Localization of a fluorescently tagged candidate centrosome protein Each group will be given a plasmid containing a cDNA that encodes a candidate centrosome protein. In the first week you will design PCR primers capable of amplifying your cDNA. In the second week you will use these primers to isolate a PCR product containing your cDNA. In the third week you will clone your cDNA into an entry vector, and amplify the entry vector in bacteria. In the fourth week you will isolate plasmid DNA from bacteria, use restriction analysis to identify the correct clone, perform a recombination-based cloning procedure to transfer your cDNA into a DsRed expression vector, and amplify the expression vector in bacteria. In the fifth week you will isolate plasmid DNA from bacteria and use restriction analysis to identify the correct expression clone. In the sixth and seventh weeks you will transfect your expression construct into HeLa cells and perform IIF to determine whether your protein localizes to centrosomes. Experiment 6: siRNA-mediated depletion of a candidate centrosome protein in HeLa cells Each group will also use their cDNA to generate small interfering RNA (siRNA) molecules for use in depleting the mRNA encoding your protein. In the fourth week you will design PCR primers that introduce T7 promoters into a portion of your cDNA. In the fifth week you will use these primers to generate a gene-specific DNA template flanked by T7 promoters, and use T7 RNA polymerase to transcribe a region of your cDNA into double stranded RNA (dsRNA). In the sixth week you will use RNAseIII to cleave this dsRNA into siRNAs, and in the seventh week you will transfect this siRNA into cells and harvest the siRNA-transfected HeLa cells. In the eighth week you will perform IIF to determine whether your candidate protein has a centrosomal function, and in the ninth week you will perform immunoblot analysis to determine whether depletion of your candidate protein altered the levels of Mps1. Maintaining and Passaging Tissue Culture Cells Starting in the sixth week, you will be given a culture of HeLa cells that have been engineered to express the centriolar protein Centrin 2 (Cetn2) that is tagged with the Green Fluorescent Protein (GFP) (HeLa GFP-Cetn2 cells, described below in Figure 14) and learn the sterile technique and cell handling procedures required to maintain the culture. You will then be required to maintain your cells and prepare them for experiments 5 and 6 as described below.


A. no detergent B.

+ Triton X-100

Analysis of Cellular Membranes and the Cytoskeleton (Experiment 1) Lectins are proteins that bind to sugar residues in a highly selective manner. Wheat germ

agglutinin (WGA) is a lectin that binds to N-acetylglucosamine (GlcNAc), a common constituent of oligosaccharides, and is often used by cell biologists to study glycoproteins. Many glycoproteins are found on the plasma membrane, but oligosaccharides are added to proteins in the endoplasmic reticulum and modified in the Golgi. Accordingly, glycoproteins must transit through the cellular membrane systems to reach the cell surface, and are also found in many locations inside the cell. The cytoskeleton is very important for cellular function, in part because it maintains the proper distribution of cellular membrane systems. Many plant and fungal toxins target cytoskeletal proteins. For example, latrunculin A from red sea sponges binds to monomeric actin (also known as globular, or G-actin), preventing microfilament assembly, while phalloidin from the death cap mushroom binds to filamentous actin (F-actin) and stabilizes microfilaments. Similarly, vincristine from periwinkle binds to tubulin dimers and prevents the assembly of microtubules, while paclitaxel from the pacific Yew tree binds to β-tubulin and stabilizes microtubules. The proper function of the mitotic spindle requires highly dynamic microtubules that can be disassembled as rapidly as they are assembled, and vincristine and paclitaxel are powerful anti-tumor agents that block the division of rapidly dividing cells.

The high affinities of natural molecules for cellular proteins make them useful tools for cell biologists. Once coupled to fluorochromes such as fluorescene (FITC, a green dye) or rhodamine (RITC, a red dye), they can be used to visualize the cellular distribution of their target proteins. We will study the distribution of cellular membrane systems, microfilaments, and microtubules using FITC-WGA, RITC-phalloidin, and FITC-paclitaxel. You will fix cells with formaldehyde, and then treat them either with or without the non-ionic detergent Triton X-100. This mild detergent dissolves the membranes, leaving the underlying cellular proteins fixed in place. When the plasma membrane is intact, FITC-WGA can access proteins present on the exterior of cells, but not those on the interior. Once membranes have been disrupted with Triton X-100, FITC-WGA, RITC-phalloidin, and FITC-paclitaxel can bind to GlcNAc-containing proteins, microfilaments, and microtubules present in the interior of the cell.

Figure 9. Schematic Representation of Experiment 1.

(A) shows a cell with an intact membrane (e.g. no Triton X-100 added), where FITC-WGA can only bind to glycoproteins (represented by a sunburst) that are present on the plasma membrane, but cannot bind to those inside the cell. Paclitaxel and phalloidin likewise cannot access the inside of the cell.

(B) shows a cell treated with Triton X-100, where FITC-WGA, FITC-paclitaxel, and RITC-phalloidin can stain glycoproteins (green), microtubules (green), and microfilaments (red) inside the cell.


A. Cellular Organization B.

+ nocodazole

Microtubules and Cellular Organization (Experiment 3) Many cytoplasmic components depend on microtubules for their proper distribution within the cell. Microtubules are inherently polarized structures with biochemically distinct ends; α-tubulin is exposed at one end of a microtubule (called the minus end) while β-tubulin is exposed at the opposite end (called the plus end) where heterodimers can be added at a much higher rate than at the minus end. Centrosomes are the primary nucleation site for microtubules, acting as microtubule organizing centers by organizing the minus ends of microtubules. This allows the plus ends to grow outward in all directions to create an inherently directional microtubule network with the minus end at centrosomes and plus ends extending toward the cell periphery. A class of proteins called microtubule motors utilized can walk along microtubules toward one end or the other to transport cargo toward or away from the centrosome. For example, proteins synthesized in the endoplasmic reticulum are sorted into vesicles that bind to minus end-directed motors, which transport the vesicles toward microtubule minus ends until they fuse with the Golgi complex. The Golgi complex itself is held in a pericentrosomal position by a balance between plus end-directed and minus end-directed motors. In the Golgi proteins are sorted into vesicles that are transported toward microtubule plus ends until they fuse with the trans-Golgi network, and vesicles from the trans-Golgi network are transported toward microtubule plus ends until they fuse with endosomes. From endosomes vesicles can be transported toward microtubule minus ends to lysozomes. At the same time, proteins internalized from the cell surface are transported toward microtubule minus ends to the endosomes, although the delivery of vesicles from endosomes to the plasma membrane does not appear to require microtubules. Accordingly, centrosomes are at the center of cellular traffic control, and disruption of the microtubule network leads to the disorganization of structures in the cytoplasm. We will examine the role of centrosomes in maintaining cell shape and organization by studying the distribution of the Golgi complex in cells that have been treated with nocodazole, a microtubule poison that like vincristine causes the depolymerization of the microtubule network. Figure 10. Schematic Representation of Experiment 3. (A) shows the role of microtubules in the positioning and communication between cellular compartments. Red arrows indicate the route of proteins newly synthesized in the endoplasmic reticulum, and black arrows indicate the route of proteins internalized from exterior or surface of the cell. (B) shows the consequences of disrupting microtubules for the organization of cellular components such as the endoplasmic reticulum, Golgi complex, and Endosomes.


Centrosomes and Centriole-associated Structures (Experiment 4) The proteomic analysis of the human centrosome identified roughly 500 proteins, and the goal of the original research projects in MG602 is to determine whether these proteins are bona fide centrosomal components. Centrosomes are highly complex, and there are many distinct environments that these proteins might be found in. The primary distinction within centrosomes is that of centrioles versus the pericentriolar matrix. However, centrioles are also very complex, and the two centrioles within a centrosome differ both temporally (Figure 11A) and structurally (Figure 11, B-D). The two centrioles have a mother-daughter relationship; the assembly of the daughter centriole was directed by the mother centriole in the previous cell cycle, and the mother centriole was assembled at least one cell cycle before its daughter (Figure 11 A). In addition, the mother centriole is distinguished from its daughter by specialized structures called appendages (Figure 11 B). During the process of centriole replication each centriole will form a structure called the cartwheel, which will serve as the template onto which the remaining centriole proteins are assembled (Figure 11, C and D). The result is that the proximal end of a centriole (the end closer to its mother) is made up of different proteins than its distal end. During mitosis the cartwheels that served as a template for the assembly of the daughter centrioles are degraded and appendages are added to the two oldest centrioles. Accordingly each daughter cell inherits an identical centrosome with at least six distinct structural environments; pericentriolar matrix, centriole wall, proximal end, distal end, maternal centriole, and centriolar appendages. We will examine the structures associated with centrosomes in cells that express a GFP-tagged version of the centriole protein Centrin 2 (GFP-Cetn2), by transfection of a variety of centrosomal proteins from various locations that are tagged with the red fluorescent protein DsRed. Figure 11. Centriole Replication and Centriole-associated Structures. (A) The generational relationship between centrioles over three rounds of replication. Existing centrioles are conserved at each round of replication, such that after replication each centrosome has a daughter centriole assembled during that cycle and a mother that can be traced back to the previous cell cycle, or even earlier depending on its lineage (e.g. the black and white centrioles can be traced back to the original centrosome at the left). (B) An electron micrograph showing the centrosome in a G1 cell, e.g. at the beginning of the cell cycle, showing the mother centriole (M) with its appendages (ap) and the daughter centriole (d). The inset shows a high magnification fluorescent micrograph of a similar centrosome showing the distal centriole protein Cetn2 in green and the appendage protein Cep170 in red. (C) Fluorescent images of centrosomes during replication showing Cetn2 in green and the cartwheel (c) protein Sas6. Top panel shows a pre-replication G1 centrosome. Middle panel shows a centrosome after formation of cartwheels (c) but before the new centrioles have been assembled. Bottom panel shows a centrosome after assembly of the new daughter centrioles (d’ and d”). (D) Schematic diagram of the fluorescent images in (C). Appendages of the maternal centriole are shown in purple, and the cartwheel is shown as a nine-spoked structure with Sas6 (red) at its hub.


Construction of a DsRed-tagged expression construct (for Experiment 5) We will use a recombination based cloning technique, developed by Invitrogen Inc., to tag your candidate centrosome proteins with the red fluorescent protein DsRed in an vector that can be used to express your DsRed-tagged proteins in human cells. The system we will use is modeled off of bacteriophage lambda, which integrates into and excises from the bacterial chromosome by recombination between sequences called att sites (Figure 12 A below). The Gateway® cloning system (Figure 12 C below) utilizes the lambda att sites to drive directional transfer a gene of interest (GOI) from a starting plasmid (called an entry vector) into one of several expression plasmids (called destination vectors). Entry vectors harbor the kanamycin bacterial resistance marker (kanr). Destination vectors harbor the ampicillin resistance marker (ampr) and the ccdB gene. Because entry vectors lack ampr and the ccdB gene is toxic to E. coli strain DH5α, neither vector can transform DH5α to ampicillin resistance. However, recombination between an entry vector and a destination vector, catalyzed by a mix of lambda recombination proteins called LR Clonase®, results in the GOI swapping places with the ccdB gene.

Entry vectors do not contain any information required to express the GOI, while destination vectors contain DNA sequences that direct the expression of the GOI, typically fused to a specific epitope tag. Therefore, recombination between an entry vector and a destination vector transfers the GOI from a universal starting vector (the entry vector) into a specialized expression vector that can transform DH5α to ampicillin resistance. The power of the Gateway® system is that once you have made an entry vector containing a GOI, it can be rapidly used to generate a virtually unlimited number of specialized vectors for the expression of variety of tagged versions in various cell types for a broad range of experiments.

We have obtained cDNAs encoding each of the candidate centrosome proteins. You will first generate a PCR product containing your cDNA that you will clone into an entry vector that is chemically coupled to DNA Topoisomerase (Figure 12B). You will then tag your candidate proteins with DsRed by using LR Clonase® to transfer your cDNA into pDEST-DsRed to create an expression clone that expresses a DsRed-tagged version of the protein encoded by your cDNA.

Figure 12. Recombination based cloning.

(A) The Lambda (λ) bacteriophage genome is circularized upon entry into the host cell to create a single attP site. The bacterial genome contains a single attB site into which λ integrates by homologous recombination to create hybrid attL and attR sites. Excision is achieved by recombination between the attL and attR sites, which regenerates the attP and attB sites.

(B) A PCR product amplified from a vector containing a Gene of Interest (GOI) can be cloned into pENTR-TOPO via the coupled Topoisomerase to generate and entry construct for the Gateway® system.

(C) The Gateway® system utilizes λ att sites to transfer a GOI between entry vectors and destination vectors, neither of which can allow E. coli strain DH5α to grow in the presence of ampicillin. Only expression vectors resulting from recombination have the Ampr gene and lack the toxic ccdB gene.


Generation of siRNA Pools (for Experiment 6) RNA interference is mediated by siRNAs - 21 nucleotide double stranded RNA molecules with single base overhangs - that direct the sequence-specific destruction of mRNAs (Figure 13 A below). siRNAs accomplish this by binding to the RNA Induced Silencing Complex (RISC), which becomes a sequence-directed ribonulcease that destroys any single stranded RNA molecules (e.g. mRNAs) that the siRNA can base pair with. This mechanism is extraordinarily specific and even a single base pair mismatch can prevent siRNA-mediated mRNA degradation. This allows researchers to introduce an siRNA into cells in order to specifically target any mRNA of interest for degradation, which ultimately results in the depletion of the protein encoded by that message.

It is therefore important to have a source of siRNAs that can be introduced into cells. The conventional source is a nucleic acid synthesis company such as Dharmacon Inc., the first company to produce and sell siRNAs. However, siRNAs have at best a 50% success rate, and several siRNAs for each gene must be screened to find an siRNA that effectively depletes the protein of interest. This approach is not cost effective when testing multiple genes. Recently, several approaches have allowed the production of siRNAs at the benchtop. In one such technique, pools of siRNAs against a single target gene are created (Figure 13 B below). This has two advantages: First, because each pool is likely to contain several highly effective siRNAs, pools are almost always more effective than a single siRNA and there is no need to test more than one pool for any given gene. Second, for the cost of synthesizing a single siRNA, a researcher can generate siRNA pools against several genes, making this approach both quicker and more cost effective when testing multiple genes. This is the approach we will use to generate siRNAs for this course.

You will generate PCR products representing a 300-500 base pair region of your cDNA. Because the PCR primers you will use include the sequence of the T7 RNA polymerase promoter, this PCR reaction will generate a product with a portion of your cDNA flanked by T7 promoters. You will use this as a template to generate dsRNA corresponding to your cDNA using the T7 RNA polymerase. You will then use RNAseIII to cleave the 300-500 base pair dsRNAs into the smaller 21 and 22 base pair siRNAs that are effective in RNA interference (Figure 13 B below). Figure 13. RNA Interference and the Generation of siRNA pools. (A) Mechanism of RNA interference. An RNAse III enzyme called Dicer processes long double stranded RNA (dsRNA) molecules into smaller molecules of 19-22 nucleotides in length called small interfering RNA (siRNA) molecules. These siRNAs bind to the RNA-Induced Silencing Complex (RISC) and activate its nuclease activity in a highly sequence-specific manner.

(B) Scheme for generation of siRNA pools.

B. A.


Guidelines for Maintaining and Passaging Tissue Culture Cells (for Experiments 5 and 6) Cell culture may appear easy at first, but any lapse in your attention can lead to contamination, and any deviation from standard culture procedures can cause your experiments to fail. For example, transfection with DsRed expression constructs becomes difficult if cells become crowded in the days before an experiment, and the effectiveness of siRNA is highly sensitive to the number of cells at the time of transfection. You will be responsible for maintaining healthy cultures for use in your experiments, and if you neglect your cell cultures or you use too many cells, your experiments may not work. However, if you follow the protocols carefully you will have no problem maintaining healthy cultures that will permit you to efficiently carry out your experiments. Sterile technique Sterile technique, also referred to as aseptic technique, is critical for maintaining healthy, uncontaminated cultures. Tissue culture medium is very rich in nutrients and easily contaminated by microorganisms in the air. The most obvious route of contamination is leaving a culture open to the air, but this is not the most common route. More commonly pipettes or media bottles are contaminated by contact with gloves or work surfaces, and the contamination is subsequently transferred into a sterile culture or the sterile contents of a media bottle.

Therefore, the most effective practices in sterile technique are sanitizing work surfaces and paying careful attention to the tips of pipettes and the mouths of media bottles. First, gloves should be worn at all times, and sterilized using a towel wetted with 70% ethanol at the beginning of work, and periodically thereafter. Second, work surfaces should be wiped with disinfectant solution followed by 70% ethanol. At any time during your work, if you suspect the work surface has become contaminated, e.g. with culture medium or cells, it should be disinfected again. Note that 70% ethanol should not be used as the initial decontaminant, because ethanol will fix the proteins in the medium to the surface rather than removing them. The proper procedure for disinfecting a contaminated surface is to first wipe the surface with a liquid disinfectant solution, and then to sterilize it with 70% ethanol. Furthermore, if at any time during your work you suspect a pipette to have come in contact with a work surface, even if you believe the surface to be sterile, the pipette should be immediately discarded, even if it contains media or cells. Similarly, if the mouth of a media bottle comes in contact with a work surface (e.g. your finger or forearm), it should be immediately disinfected using a towel wetted with 70% ethanol. Note that care should be taken to avoid the introduction ethanol into the medium, and to NEVER wipe a media bottle with disinfectant; too much ethanol and any amount of disinfectant can kill your cells.

Of course, the air in the lab is also a potential source of contamination. To minimize exposure to room air, bottles and plates should be kept closed as much as possible and tilted before opening to present the lowest possible cross section to air and falling particles while still permitting the required operation, e.g. insertion of a pipette. Care should be taken not to exaggerate this process or media can be sloshed from culture plates, or come into contact with the lip of the media bottle, a hot spot for contamination as described above.

Briefly flaming the surfaces of pipettes and bottles is the final measure that completes good sterile technique. Pipettes can be flamed before use (this is both unnecessary and unwise for individually wrapped disposable plastic pipettes), and bottles can be flamed after removing a portion of media with a pipette. Note that the most important effect of flaming a pipette or bottle is to suck the air out. That is, the intent of flaming is not to kill contaminants that may have landed on the surface, but rather to remove any airborne contaminants that may have entered the pipette or bottle.

Note that lab coats are another potential source of contamination. Particles can fall into your culture from any object that “hovers” over the plate. Lab coats are difficult to keep clean and have loose floppy sleeves. Keep this in mind as you perform your tissue culture. Tissue Culture Waste Tissue culture medium must be decontaminated with bleach before it can be discarded. Therefore, discard all used media and washes into a beaker for transfer into the waste bucket at the front of the room. At the end of each lab period the waste will be decontaminated and disposed of. Pipettes must also be decontaminated properly. Glass pipettes must be placed in the provided pipette dumps (gently to avoid damage), whereas disposable Pasteur and plastic pipettes must be placed in the biohazardous waste container.


Passaging Cells: To passage cells, also called splitting cells, means transferring cells from one culture to a fresh

culture at a lower cell density. Most tissue culture cells are adherent and only grow when they are attached to a surface. Thus, a given number of cells will be at the same density regardless of how much medium is in the dish, and cell density is defined as cells per unit growth area. It is critical to note that the terminology used for passaging cells (e.g 1:5 or 1:10) refers to the fraction of the original culture that will be used to seed a new culture, rather than to the volume used to transfer the cells or the volume they are transferred into. As an example to consider, 106 cells transferred to a 10 cm dish in 10 ml of medium will be at the same cell density as 106 cells transferred to a 10 cm dish in 50 ml of medium. The second cell transfer is 5-fold less concentrated at the time of plating, but the cells from both solutions will settle onto the cell surface of the dish and number of cells per unit area will be the same on both dishes.

The use of adherent cells adds one additional constraint to passaging cells; the cells must be removed from the surface of the dish before they can be passaged. This is accomplished through an enzymatic cleavage of the cell surface proteins that contact the dish. There are several such “cell dissociation” reagents, and we will use an enzyme called trypsin.

In the typical cell culture experiment cells are grown in 10 ml of medium in a 10 cm tissue culture (TC) dish. To passage cells, the old media is removed, cells are rinsed to remove residual media, and a small amount of cell dissociation reagent is placed over the cells. Once the cells have dissociated from the plate, they are resuspended in fresh medium. With very few exceptions, you will always resuspend cells in a total volume of 10 ml to simplify the calculation of the volume you need to transfer. To split cells at 1:10 means to transfer one tenth of the cells from the original plate into a fresh plate of the same size. Because you will resuspend the cells in 10 ml and transfer them into 10 ml of medium, this becomes a simple dilution problem. However, if you change either the resuspension volume or the volume on the new plate, it is important to remember that you want to transfer one tenth of the cells in the original dish (or one fifth for a 1:5 split, etc). A common mistake is to double the amount of medium without changing the number of cells to achieve a higher passage ratio. However, if you resuspend cells in 10 ml and transfer 1 ml to a new dish with 20 ml of medium, it is still a 1:10 split because you added one tenth of the cells from the original dish.

The HeLa cells you will be culturing are robust and grow very predictably. For HeLa cells, a 1:2.5 split will be ready to split again the next day, a 1:5 split will be ready in two days, a 1:10 split will be ready in 3 days, and a 1:20 split will be ready in 4 days. To achieve these passage ratios, or splits, first resuspend the tyrpsinzed cells in 10 ml of total volume. Then, for a 1:2.5 split, transfer 4 ml to the fresh plate; for a 1:5 split transfer 2 ml to the fresh plate; for a 1:10 split transfer 1 ml to the fresh plate; for a 1:20 split transfer 0.5 ml to the fresh plate. DO NOT DEVIATE FROM THIS FORMAT! Keep in mind that it is the number of cells transferred that is critical, NOT the final volume on the target plate. Cell Passages For This Course: Tu 5/4 Passage cells at 1:2.5, 1:5, 1:10, and 1:20 Th 5/6 Passage cells at 1:10, and 1:20 (NOTE: You MUST have a plate of cells ready on Monday, May 10)

Mo 5/10 Come in after noon to passage cells for Tuesday passage at 1:2.5 and 1:10 as normal for future experiments

plate cells in 6-well dish for transfection experiment Tu 5/11 Passage cells at 1:5 and 1:10 Th 5/13 Passage cells at 1:10 and 1:20 (NOTE: Cells MUST be ready on Monday, May 17) Mo 5/17 Come in after noon to passage cells for maintenance and for transfection experiments passage at 1:2.5 and 1:10 as normal in case you need to repeat any experiments

plate cells in 6-well dish at two different densities for transfection experiment


HeLa GFP-Cetn2 Cells (for Experiments 4, 5, and 6)

Figure 14. HeLa-GFPCetn2 Cells. This cell line will be used for Experiments 4, 5 and 6, the fluorescent analysis of centrosomes in HeLa cells. These cells that inducibly expresses GFP-Cetn2 were created by your TA, Julia Yang, as part of her PhD thesis research. These cells harbor one construct that constitutively expresses the Tetracycline Repressor protein (Tet R) under the control of the CMV early promoter, and a second construct that expresses GFP-tagged Centrin 2 (GFP-Cetn2) under the control of the Tetracycline Operator (TetO2). TetR is a dimeric sequence-specific transcriptional repressor, and TetO2 contains a tandem repeat of the TetR binding site. TetR expression therefore results in repression of genes under the control of TetO2. However, the ability of TetR to repress transcription is regulated by tetracycline (tet). Tet binds to TetR, causing a conformational change that prevents DNA binding and releases TetR from TetO2. Accordingly, TetO2-regulated genes can only be expressed in the presence of tet when the TetR cannot repress transcription.


Laboratory Exercises: On most days you will be multitasking, and working on two or even three experiments at once. While it is important to give you a complete list of the steps for each experiment, this type of protocol does not tell you how to integrate multiple protocols into a single workflow. Therefore, there is an entry for each day that is divided into two sections. The first section briefly outlines the workflow, indicating how to make one single workflow out of multiple different experiments. The second section provides detailed descriptions of each experimental protocol. Neither section alone will be sufficient to tell you exactly what to do at any given time: The workflow tells you how to switch back and forth between protocols to achieve a logical work flow, but does not tell you how to perform any single step. In contrast, the experimental protocols tell you exactly what to do at each step, but do not give you any indication of when to switch between protocols. However, together the workflow and protocols should keep you moving forward in a timely and efficient manner.


Tuesday, March 30 Today you will be given RPE1 (Retinal Pigment Epithelial cells) that have been grown on

coverslips. You will fix these cells with formaldehyde, treat them either with or without the detergent Triton X-100 to dissolve the plasma membrane, and then stain them with FITC-WGA, FITC-paclitaxel, and RITC-phalloidin.

Workflow: A. Fix Cells I. Steps 1-4 B. Extract Cells with Triton X-100 II. Steps 1-3 C. Stain Cells

III. Steps 1-3 D. Mount Coverslips IV. Step 1-3 Experimental Protocols: I. Fix Cells (Exp 1)

(see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol) 1) Transfer 4 coverslips into a 24 well plate 2) Add 0.15 ml 4% formaldehyde to each well

- wear gloves, formaldehyde is toxic! 3) incubate 10 minutes at room temperature 4) Wash coverslips five times with ~0.5 ml non-sterile PBS

- discard formaldehyde and first PBS wash into a labeled waste beaker - discard subsequent washes as standard waste

II. Extract Cells with Triton X-100 (Exp 1) 1) Extract half of your coverslips a) add 0.15 ml 0.2% Triton X-100 in PBS to coverslips #1 and 2 b) add 0.15 ml PBS to coverslips #3 and 4 2) incubate 15 minutes at room temperature 3) Remove contents from each well and wash coverslips once in PBS III. Stain Cells (Exp 1) 1) Add the following mixes to each coverslip: a) add 0.15 ml FITC-WGA + RITC-phalloidin mix to coverslips #1 and 3 b) add 0.15 ml FITC-paclitaxel + RITC-phalloidin mix to coverslips #2 and 4 2) incubate for 15 minutes at room temperature 3) wash coverslips twice with PBS IV. Mount Coverslips (Exp 1)

(see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol) 1) Prepare slides

a) label the frosted end of a slide with initials and date b) place four drops of mounting medium in a line on the labeled slide

2) Transfer coverslips onto slides a) invert each coverslip onto a drop of mounting medium b) remove excess mounting medium

3) Seal coverslips a) once coverslips are dry, seal around edges of coverslips with nail polish

Store the prepared slides in a microscope slide box for examination on Thursday.


Thursday, April 1 Today will consist of a microscope tutorial that introduces some of the theory behind fluorescence microscopy and teaches you the basics of using the Olympus BX-41 upright fluorescent microscope, followed by time on the microscopes and a homework assignment. For the homework assignment you will design the primers that will be used for PCR reactions to amplify a cDNA encoding a candidate centrosome protein. You can work on the homework in class or over the weekend, but it is due by the beginning of class on Tuesday, April 6. We will order primers based on the sequences you designed for use in PCR on Thursday, April 8.

The BX-41 fluorescence microscopes we will use in the course are very sophisticated, and hence expensive instruments. Accordingly we only have five microscopes available and it will not be possible for everyone to use the microscopes at the same time. After the microscope tutorial, half of the groups will look at their slides from Tuesday while half learn about the homework assignment. Halfway through class the groups will switch.


Tuesday, April 6 Remember that your homework assignments (primer design) are due today by the start of class. You can either email your sequences to your TAs, or hand in a print out, but no credit will be given for sequences handed in after the start of class.

Today you will be given RPE1 cells for two different experiments. In the first experiment you will transfect cells with a construct that expresses GFP-tagged Tubulin. In the second experiment you will fix cells as you did previously and perform Indirect Immunofluorescence (IIF) to look at the relationship between centrosomes and other cellular structures. Workflow: A. Fix Cells I. Steps 1-4 B. Perform IIF II. Step 1 C. Transfect Cells III. Steps 1-6 D. Perform IIF II. Steps 2-6 Experimental Protocols: I. Fix Cells (Exp 2)

(see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol) 1) Transfer coverslips into a 24 well dish 2) Add 0.15 ml F/T to each well and incubate 10 minutes at room temperature 3) Wash coverslips five times with ~0.5 ml non-sterile PBS


II. Indirect Immunofluorescence (IIF) (Exp 2a) (see Protocol #3 “Indirect Immunofluorescence (IIF) Analysis” on page 62 for detailed protocol) 1) Block non-specific protein binding sites a) 0.25 ml of IIF Blocking buffer

b) incubate 60 minutes at room temperature in the dark* 2) Add primary antibodies

a) remove blocking buffer and add 0.15 ml primary antibody mix as follows: - coverslip #1 and 3: mouse anti-M6PR + rabbit anti-γTubulin - coverslip #2 and 4: mouse anti-αTubulin + rabbit anti-γTubulin NOTE: one lab partner should processes coverslips #1 and 2, the other should process #3 and 4

b) incubate 60 minutes in the dark

3) Wash cells 5 times with non-sterile PBS - use a pipettor to remove antibody mixes, changing tips to avoid cross contamination - discard antibodies and washes as standard waste

4) Add secondary antibodies a) remove final PBS wash and add 0.15 ml secondary antibody mix to each well b) incubate 60 minutes at room temperature in the dark


5) Wash coverslips 5 times with ~0.5 ml PBS a) remove and discard secondary antibody solutions

- use a pipettor to remove antibodies, changing tips to avoid cross contamination - antibodies can be discarded as standard waste

6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol) * “In the dark” is achieved by placing dish into a cubby under your bench † use non-sterile PBS for IIF, do not use the same sterile PBS you use for culture III. Transfect cells (Exp 2b)

(see Protocol #4: “Effectine Transfection of Plasmid DNA” on page 63 for detailed protocol) 1) Sterilize your bench tops 2) Mix Transfection Reagents: a) Add the following IN ORDER to a microcentrifuge tube

138 µl Buffer EC 4 µl Plasmid DNA (~1 µg) 8 µl Enhancer solution

b) Mix by vortexing for 2 seconds c) Incubate 5 minutes at room temperature d) Store remaining Effectine, Buffer EC, and Enhancer at 4 °C for later use

3) Make transfection complexes

a) add 25 µl Effectene Reagent b) Mix by vortexing 10 seconds c) Incubate 10 minutes at room temperature

4) Wash cells and add fresh medium (during the 10 min incubation of step 3) a) follow sterile technique practices b) remove medium and wash cells with 4.0 ml sterile PBS c) remove PBS wash and add 3.0 ml fresh, pre-warmed medium

5) Add transfection mix to cells a) add to mix transfection complexes created in step 3 with 1.0 ml fresh medium b) transfer transfection mixture dropwise onto cells

6) Return cells to the incubator


Thursday, April 8 Today you will examine your IIF from Tuesday, process the cells you transfected on Tuesday for IIF, and set up PCR reactions using the primers you designed and ordered. All groups will start by setting up their PCR reactions. However, because there are not enough microscopes we will split the groups in half again. The group that used the microscopes first last week will start processing their transfected cells for IIF while the other groups examine Tuesday’s IIF on the microscopes. The groups will switch halfway through (this will hopefully coincide with the completion of the PCR reactions so that we can load the PCR reactions on an agarose gel at the switch point); the groups that were using the microscopes will begin processing their transfected cells for IIF while the other groups use the microscopes to analyze Tuesday’s experiment. Workflow: A. Set up PCR reactions I. Steps 1-3 B. Fix transfected cells II. Steps 1-4 C. Perform IIF III. Steps 2-5 D. Analyze PCR reactions on a gel I. Step 4 E. Set up TOPO cloning Reactions IV. Steps 1-5 Experimental Protocols: I. Set up PCR reactions (Exp 5): NOTE: Be extremely careful at this point not to cross contaminate the stock tubes 1) Combine the following in a microcentrifuge tube on ice

Reaction Mix 90 µl primer mix 4 µl template plasmid 2 µl

(~25 ng of pcDNA-CLIC1, -FAM161A, -FKBP3, -VDAC1, or -VDAC2) 2) Transfer 48 µl to each of two 0.2 ml PCR tubes

a) add 2 µl sterile water to one tube (“negative control”) b) add 2 µl of Platinum Taq solution to the other tube (“experimental”) c) mix gently with pipettor

3) Take your PCR reactions to the TAs who will run the PCR machine Note the “Address” of your PCR reaction for later retrieval 4) Check the reactions on a gel

a) TAs will distribute PCR reactions once completed b) Place two ~2 µl spots of 10x loading dye onto parafilm

c) pipette 5 µl of each PCR reactions directly onto loading dye and mix d) load mixture into well Note position on gel of your PCR reaction e) Visualize gel on the Dark Reader f) TAs will run gel and take a picture


II. Fix Cells (Exp 2b) (see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol)

1) Transfer coverslips into a 24 well dish 2) Add 0.15 ml F/T to each well and incubate 10 minutes at room temperature 3) Wash coverslips five times with ~0.5 ml non-sterile PBS


III. Indirect Immunofluorescence (IIF) (Exp 2b) (see Protocol #3 “Indirect Immunofluorescence (IIF) Analysis” on page 62 for detailed protocol) 1) Add 0.25 ml of IIF Blocking buffer, incubate 60 minutes at room temperature in the dark* 2) Remove blocking buffer, add 0.15 ml antibody mixes as follows, and incubate 60 minutes in the dark - coverslip #1: mouse anti-M6PR - coverslip #2: mouse anti-γTubulin - coverslip #3: mouse anti-mitochondria - coverslip #4: phalloidin 3) Remove 1° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 4) Remove final PBS wash, add 0.15 ml secondary antibody, and incubate 60 minutes in the dark 5) Remove 2° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol) IV. Set up TOPO-TA cloning reactions (Exp 5) 1) Mix PCR product, pENTR-SD/D-TOPO, buffer, and enough water for a final volume of 6.0 µl 0.5-4.0 µl PCR product ( 1.0 µl vector 1.0 µl salt solution 0-3.5 µl sterile water (6.0 µl final volume) 2) Mix gently 3) Incubate 5 minutes at room temperature 4) Place reaction on ice 5) store reaction at 4 °C


Tuesday, April 13 Today you will be working on two experiments. You will be treating RPE1 cells with nocodazole to disrupt microtubules in order to study the role of the centrosome in cellular organization, and you will be cloning the PCR products you generated last week into an entry vector in order to generate a DsRed-tagged version of a candidate centrosome protein. Workflow: A. Treat Cells with Nocodazole I. Steps 1-5 C. Fix Cells II. Steps 1-3 D. Transform Bacteria IV. Steps 1-4 E. Perform IIF III. Steps 1-6 Experimental Protocols: I. Treat Cells With Nocodazole (Exp 3) 1) Sterilize your benchtop 2) Take one dish with four coverslips from the incubator 3) Add nocodazole to 200 ng/ml 4) Return cells to incubator 5) Incubate 2 hours II. Fix Nocodazole-treated Cells (Exp 3)

(see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol) 1) Transfer coverslips into a 24 well dish 2) Add 0.15 ml F/T to each well and incubate 10 minutes at room temperature 3) Wash coverslips five times with ~0.5 ml non-sterile PBS


III. Indirect Immunofluorescence (IIF) (Exp 3) (see Protocol #3 “Indirect Immunofluorescence (IIF) Analysis” on page 62 for detailed protocol) 1) Add 0.25 ml of IIF Blocking buffer, incubate 60 minutes at room temperature in the dark* 2) Remove blocking buffer, add 0.15 ml antibody mixes as follows, and incubate 60 minutes in the dark - coverslip #1: mouse anti-M6PR - coverslip #2: mouse anti-αTubulin - coverslip #3 rabbit anti-γTubulin - coverslip #4: phalloidin 3) Remove 1° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 4) Remove final PBS wash, add 0.15 ml secondary antibody, and incubate 60 minutes in the dark 5) Remove 2° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol)


IV. Transform pENTR-TOPO cloning reactions into Bacteria (Exp 5) (see Protocol #5: “Bacterial Transformation” on page 64 for more details)

- TAs will thaw competent TOP-10 cells on ice, and dispense them into individual tubes - each group should take one tube and place it IMMEDIATELY on ice - it is important to keep the cells as cold as possible until the heat shock in step 2 NOTE: This is our first of two bacterial transformations. It is recommended that one student perform this transformation while the other student observes, so that the observer can perform the procedure next time. Label your tube(s) with your initials and a description of the sample (e.g. “+” and “-“) 1) Pipet 1 µl of the pENTR-TOPO cloning reaction to the TOP-10 cells

a. gently mix sample and cells b. incubate 10 minutes on ice

2) Heat shock the mixture of cells and DNA in the 42 °C water bath for EXACTLY 45 seconds a. return tubes to ice for 1 minute

3) Add 250 µl SOC medium to the mixture of cells and DNA tube a. allow cells to recover by incubation for 60 minutes at 37 °C with shaking - use sterile technique, SOC is extremely rich medium

4) Plate entire transformation reaction a. pellet bacteria in microfuge

- 1,000 g for 30 seconds b. remove 200 µl medium (using sterile technique) c. resuspend bacteria in remaining medium d. spot bacteria onto LB plate containing Kanamycin e. spread the cells evenly using a sterile spreader to distribute the liquid f. incubate plates in 37 °C bacterial incubator overnight

Note:

NEVER incubate bacterial cells in the same incubator with tissue culture cells! NEVER handle tissue culture cells immediately after working with bacteria! Wash hands with soap and water first.


Thursday, April 15 Today you will be examining the last two experiments you performed on the microscope, and amplifying individual clones from your bacterial transformations in order to isolate the plasmid DNA. We will split into groups again. Today, the group that used the microscopes first last week will start with picking bacterial colonies, and the group that used the microscopes second last week will start on the microscopes and discuss the homework assignment; designing PCR primers to flank your cDNA with T7 promoters in order to generate siRNA against the mRNA encoding your candidate protein. Workflow: A. Pick Bacterial Colonies I. Steps 1-5 B. Homework Experimental Protocols: I. Pick Bacterial Colonies (Exp 5) 1) Dispense 3.0 ml LB+Kan liquid medium into each of three sterile culture tubes 2) Label your tubes 1-3 or 4-6 with your initials 3) using a P200 pipettor, stab a single colony with a yellow pipette tip -try to get the colony on the side of the tip, not the inside of the tip 4) eject the pipette tip into a culture tube -make sure tip is immersed in liquid 5) repeat for two more colonies, transferring each tip to a fresh culture tube NOTE: For each group, each student should pick 3 colonies for a total of 6 colonies per group


Tuesday, April 20

Today you will be given HeLa GFP-Cetn2 cells (the same cell line that you will be culturing in the second half of the course), which you will transfect with constructs expressing DsRed-tagged centrosome proteins that represent a variety of regions within the centrosome. You will also isolate plasmid DNA from the bacterial colonies you amplified last week. Workflow: A. Transfect HeLa GFP-Cetn2 cells I. Step 1-6 B. Isolate Plasmid DNA II. Steps 1-13 C. Set up Restriction Digestions III. Steps 1-8 Experimental Protocols: I. Transfect HeLa GFP-Cetn2 cells (Exp 4)

(see Protocol #4: “Effectine Transfection of Plasmid DNA” on page 63 for detailed protocol) 1) Sterilize your bench tops 2) Combine Transfection Reagents IN ORDER, mix by vortexing for 2 seconds, and incubate 5 minutes

58.4 µl Buffer EC 1 µl Plasmid DNA (~0.2 µg) 1.6 µl Enhancer solution

b) Prepare four separate mixes for pDsRed, and pDsRed-TubGCP3, -Sas6, and -ODF2 c) Store remaining Effectine, Buffer EC, and Enhancer at 4 °C for later use

3) Add 5 µl Effectene Reagent, mix by vortexing 10 seconds, and incubate 10 minutes 4) During the 10 minute incubation of step 3:

a) using sterile technique, transfer 4 coverslips to individual wells of a sterile 24-well dish b) Wash cells with 0.5 ml sterile PBS c) remove wash and add 0.25 ml fresh pre-warmed medium

5) Add 0.2 ml medium to transfection components, mix, and transfer dropwise onto cells 6) Return cells to the incubator II. Isolate Plasmid DNA from Amplified Bacteria (Exp 5)

(see Protocol #6: “Plasmid “Miniprep” DNA Isolation” on page 65 for detailed protocol) 1) Retrieve your bacterial cultures from 4° C 2) Each group member will perform three “minipreps”

a) each group will perform a total of six minipreps 3) Using a P1000 pipettor, transfer 1.5 ml of culture to a microcentrifuge tube a) avoid aspirating liquid into pipette barrel b) transfer 0.75 ml twice using the same pipette tip c) change pipette tips between cultures 4) Pellet bacteria a) spin in microcentrifuge at half speed for 2 minutes 5) Remove and discard supernatant into a waste beaker 6) Resuspend bacterial pellet

a) add 250 µl Buffer P1 +RNAse A b) respuspend by vortexing


7) Lyse bacteria a) add 250 µl Buffer P2

b) mix by gently inverting 2-3 times c) do not vortex d) incubate no longer than 5 minutes at room temperature 8) Neutralize and precipitate genomic DNA and cell debris

a) by adding 350 µl Buffer N3 b) mix by inverting 5-6 times c) do not vortex 9) Pellet genomic DNA and cell debris

a) spin samples in microcentrifuge at full speed for 10 minutes 10) Bind plasmid DNA onto purification column a) place a column into a collection tube

b) pour supernatant onto the column c) spin columns in microcentrifuge at full speed for 1 minute d) discard supernatant 11) Wash plasmid DNA a) add 0.75 ml Buffer PE to column b) spin columns in microcentrifuge at full speed for 1 minute d) discard supernatant 12) Dry column a) return column to collection tube and spin 1 minute 13) Elute plasmid DNA a) place columns in microcentrifuge tubes b) add 50 µl Buffer EB directly onto column bed c) DO NOT TOUCH COLUMN BED WITH PIPET TIP! d) spin columns in microcentrifuge at full speed for 1 minute e) discard column NOTE: In this step the plasmid DNA is released from the column into the elution buffer and collected by

centrifugation. This collected plasmid DNA is referred to as a “miniprep.” III. Restriction Digestion (Exp 5) 1) Dispense 2 µl of each miniprep into a separate clean microcentrifuge tube

a) this will create six tubes labeled 1-6, each with an individual plasmid 2) Make a restriction digestion master mix a) Add the following to a clean microcentrifuge tube

master component mix (vol per digestion)

10X Restriction Enzyme Buffer 7.0 µl (1 µl) Restriction Enzyme 1.75 µl (0.25 µl) Water 47.25 µl (6.75 µl)

3) Mix, and dispense 8 µl of Master Mix to each of the tubes prepared in step 1 a) each tube will have a total of 10 µl, or 2 µl DNA plus 8 µl master mix b) this will create one restriction digest for each plasmid you prepared above 4) mix by pipetting or finger flick 5) incubate at 37 °C for 1-2 hours 6) add 2 µl 10X Blue Juice to each reaction 7) Load samples onto an agarose gel a) each group will load 7 lanes; - one lane of markers - one lane for each digestion b) note which lanes your samples were loaded in 8) The TAs will take a picture of the gel for you


Thursday, April 22 Today you will be fixing the cells you transfected on Tuesday, and performing recombination-based cloning to transfer the cDNA you cloned earlier into an expression vector with a fluorescent tag. We will also discuss the use of comparative genomics in the analysis of centrosomal proteins, and for homework you will be assigned the task of identifying homologues of centriole proteins identified in algae. Workflow: A. Fix and Stain Transfected Cells I. Steps 1-6 B. Gateway® Recombination reaction II. Steps 1-5 C. Bacterial Transformations III. Steps 1-4 D. Homework I. Fix Transfected Cells and Counter Stain Nuclei with Hoechst (Exp 4)

(see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol) 1) Transfer coverslips into a 24 well dish 2) Add 0.15 ml F to each well and incubate 10 minutes at room temperature in the dark 3) Wash coverslips five times with ~0.5 ml non-sterile PBS


4) Add 0.15 ml Hoechst to cells, and incubate 15 minutes 5) Wash cells twice with PBS 6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol) II. Gateway® Recombination reaction (Exp 5) 1) Set up recombination reactions by mixing the following in a microcentrifuge tube on ice: 4 µl entry vector + water (~300 ng of pENTR-GOI + enough water for 4 µl total) 4 µl destination vector (300 ng of pDEST-DsRed) 8 µl 5x LR Clonase® Buffer

16 µl TE buffer 2) Split reaction in half by transferring 16 µl of the mix to a fresh tube a) add 2 µl LR Clonase® to one tube (“experimental”) - TAs will have Clonase® in an ice bucket

b) add 2 µl water to the other tube (“negative control”) 3) Incubate both tubes1 hour at room temperature 4) Add 2 µl Proteinase K to each tube 5) Incubate at 37 °C for 10 min III. Transform Gateway® Recombination reactions into Bacteria (Exp 5)

(see Protocol #5: “Bacterial Transformation” on page 64 for more details) NOTE: The student who observed the procedure last time should perform the transformations this time. 1) Place two tubes of DH5α bacterial cells IMMEDIATELY on ice a) label tubes with your initials and “+” or “-” (or some other appropriate description)

b) pipet 1 µl “experimental” sample into one tube, and 1 µl “negative control” into the other c) mix gently and incubate 10 minutes on ice 2) Heat shock at 42 °C water bath for EXACTLY 45 seconds and return tubes to ice for 1 minute 3) Add 250 µl SOC medium to each tube and incubate 60 minutes at 37 °C with shaking 4) Spread 50 µl of the transformation mixture onto separate antibiotic containing plates 5) Incubate plates overnight in the 37 °C bacterial incubator

NEVER incubate bacterial cells in the same incubator with tissue culture cells!


Tuesday, April 27 Today you will set up PCR reactions as the first step in the production of siRNA, pick bacterial

colonies to amplify your DsRed expression constructs, and examine HeLa GFP-Cetn2 cells that you transfected with DsRed-tagged centrosome proteins. After setting up PCR, those groups that used the microscopes first last week will pick bacterial colonies and discuss the experiments we have performed thus far while the other groups examine their DsRed-transfected HeLa GFP-Cetn2 cells on the microscopes. Halfway through we will switch (this will hopefully coincide with the completion of the PCR reactions so that we can load the PCR reactions on an agarose gel at the switch point); the groups that were using the microscopes will pick minipreps and have a group discussion while the other groups use the microscopes to analyze Tuesday’s experiment. Workflow: A. Set up PCR reactions I. Steps 1-4 B. Examine PCR reactions on agarose gels I. Step 5 C. Pick Bacterial Colonies IV. Steps 1-5 Experimental Protocols: I. Set up PCR reactions (Exp 6): NOTE: Be extremely careful at this point not to cross contaminate the stock tubes 1) Combine the following in a microcentrifuge tube on ice

Reaction Mix 90 µl primer mix 4 µl template plasmid 2 µl

(~25 ng of pENTR-CLIC1, -FAM161a, -FKBP3, -VDAC1, or -VDAC2) 2) Transfer 48 µl to each of two 0.2 ml PCR tubes

a) add 2 µl sterile water to one tube (“negative control”) b) add 2 µl of Platinum Taq solution to the other tube (“experimental”) c) mix gently with pipettor

3) Take your PCR reactions to the TAs who will run the PCR machine Note the “Address” of your PCR reaction for later retrieval 4) Check the reactions on a gel

a) TAs will distribute PCR reactions once completed b) Place two ~2 µl spots of 10x loading dye onto parafilm

c) pipette 5 µl of each PCR reactions directly onto loading dye and mix d) load mixture into well Note position on gel of your PCR reaction e) Visualize gel on the Dark Reader f) TAs will run gel and take a picture

5) Save the remainder or your PCR reactions at 4 °C

IV. Pick Bacterial Colonies (Exp 5) 1) Dispense 3.0 ml LB+Amp liquid medium into each of three sterile culture tubes 2) Label your tubes 1-3 or 4-6 with your initials 3) using a P200 pipettor, stab a single colony with a yellow pipette tip -try to get the colony on the side of the tip, not the inside of the tip 4) eject the pipette tip into a culture tube -make sure tip is immersed in liquid 5) repeat for two more colonies, transferring each tip to a fresh culture tube NOTE: For each group, each student should pick 3 colonies for a total of 6 colonies per group


Thursday, April 29 The midterm exam will be given today. You will be given one hour for the exam. After the exam you will set up an in vitro transcription reaction for siRNA production using your PCR product as a template, and isolate plasmid DNA from your amplified bacterial colonies. Workflow: A. Midterm Exam B. Set up in vitro transcription II. Steps 1-3 C. Isolate Plasmid DNA IV. D. Set up Restriction Digestion V. Steps 1-8 Experimental Protocols: I. Set up transcription reactions (Exp 6): 1) Combine the following in a microcentrifuge tube IN ORDER AND AT ROOM TEMPERATURE

PCR product 2 µl or 8 µl* Nuclease Free Water 6 µl or 0 µl* Reaction Buffer / NTP Mix 10 µl (contains 2 µl 10x T7 Reaction Buffer and 2 µl each NTP) T7 Enzyme Mix 2 µl

* Exact volumes to be used will be determined by how well your PCR reaction worked use 2 µl PCR + 6 µl water if PCR worked well, if not use 8 µl PCR and 0 µl water

2) Mix gently by finger flicking 3) Incubate Overnight at 37 °C NOTE: TAs will perform steps 4-6 for you tomorrow morning 4) Add the following to the transcription reaction: Nuclease Free Water 21 µl 10x siRNA Digestion mix 9 µl (contains 2 µl RNAseA, 2 µl DNAseI, 5 µl 10x buffer) 5) Incubate 1 hour at 37 °C a) DO NOT INCUBATE LONGER THAN 1 HOUR! 6) Store reactions at –20 °C II. Isolate Plasmid DNA from Amplified Bacteria (Exp 5) (see Protocol #6: “Plasmid “Miniprep” DNA Isolation” on page 65 for detailed protocol) - Each group member will perform three minipreps for a total of six for each group - Perform minipreps as done on April 20, and as described on page 65 1) Retrieve your bacterial cultures from 4° C, a) resuspend bacteria by vortexing b) transfer 1.5 ml of bacterial suspension to microcentrifuge tubes c) pellet bacteria in microcentrifuge tubes by spinning 2 minutes at half speed


2) Add 250 µl Buffer P1 a) vortex until bacterial pellet is completely resuspended 3) Lyse bacteria a) add 250 µl Buffer P2 b) invert 2-3 times c) incubate no longer than 5 minutes 4) Neutralize lysate a) add 350 µl Buffer N3 b) invert 5-6 times 5) Pellet genomic DNA and cell debris in microcentrifuge at full speed for 10 minutes 6) Bind plasmid DNA by spinning supernatant through purification column at full speed for 1 minute

a) collect flow through in a collection tube and discard 7) Wash column with 0.75 ml Buffer PE at full speed for 1 minute

a) collect flow through in a collection tube and discard 8) Dry column at full speed for 1 minute

a) collect residual liquid in a collection tube and discard 9) Transfer columns to clean, labeled microcentrifuge tubes 10) Elute plasmid DNA by spinning 50 µl Buffer EB through column at full speed for 1 minute a) add directly onto column bed b) DO NOT TOUCH COLUMN BED WITH PIPET TIP! V. Restriction Digestion (Exp 5) 1) Dispense 2 µl of each miniprep into a separate clean microcentrifuge tube

a) this will create six tubes labeled 1-6, each with an individual plasmid 2) Make a restriction digestion master mix a) Add the following to a clean microcentrifuge tube

master component mix (vol per digestion)

10X Restriction Enzyme Buffer 7.0 µl (1 µl) Restriction Enzyme 1.75 µl (0.25 µl) Water 47.25 µl (6.75 µl)

3) Mix, and dispense 8 µl of Master Mix to each of the tubes prepared in step 1 a) each tube will have a total of 10 µl, or 2 µl DNA plus 8 µl master mix b) this will create one restriction digest for each plasmid you prepared above 4) mix by pipetting or finger flick 5) incubate at 37 °C for 1-2 hours 6) add 2 µl 10X Blue Juice to each reaction 7) Load samples onto an agarose gel a) each group will load 7 lanes; - one lane of markers - one lane for each digestion b) note which lanes your samples were loaded in 8) The TAs will take a picture of the gel for you


Tuesday, May 4 Today you will purify the double stranded RNA (dsRNA) that you produced in the last lab session, cleave the dsRNA into siRNA, and purify the siRNA. While you are processing your RNA samples, you will also be learning to culture cells. You will be given HeLa GFP-Cetn2 cells, and you will maintain your own cultures for the remaining experiments in the course. Workflow: A. Prepare Medium for Tissue Culture I. Steps 1-3 A. Purify dsRNA I. Steps 1-10 B. RNAseIII Cleavage of dsRNA II. Steps 1-3 B. Examine HeLa Cells II. Steps 1-3 C. Passage HeLa Cells

III. Steps 1-8 D. Purify siRNA II. Steps 1-5 Experimental Protocols: Use sterile technique at all times when handling tissue culture cells and media I. Prepare Tissue Culture Medium (Exp 5, 6)

1) Put on gloves and sterilize your bench 2) For each group of two students, prepare the following:

- four 10 cm TC dishes - four 10 ml sterile serological pipettes - two sterile Berol pipettes

- one bottle of PBS - one bottle of tissue culture medium - one tube of trypsin

3) Place the tissue culture medium and trypsin in the water bath for 10-15 minutes II Examine the cells culture under the microscope (Exp 5, 6)

1) Each group of two students will take one plate of HeLa GFP-Cetn2 cells labeled 1:5 2) Compare your plate (1:5) to the crowded (1:2) and sparse (1:10) plates 3) Note the appearance and color of the medium and the amount of debris a) keep in mind that tomorrow the 1:10 plate will look like your 1:5 plate looks today

b) the medium will be deep red or pink for a healthy culture c) the medium will be orange or yellow for an overcrowded or dying culture, respectively d) there will be only a small amount of debris (dead or dying cells) in a healthy culture e) a larger amount of debris indicates a crowded or unhealthy culture

III. Passage your HeLa GFP-Cetn2 cells (Exp 5, 6)

1) Retrieve medium and trypsin a) wipe down with 70% ethanol

2) Mix the medium by gently swirling a) avoid sloshing or bubbles

3) Transfer the medium from your plate into a waste beaker using a 10 ml pipette a) Tilt the plate for ~1 minute, remove residual media with a Pasteur pipette - NEVER insert the same pipette into your plate twice

4) Pipette 10 ml of sterile PBS into your dish, and gently wash cells a) remove the used PBS and residual as in step 3 above

5) Transfer 0.5 ml of trypsin into your dish, and coat cells with gentle rocking


6) Place your cells back in the incubator for 2 minutes 7) While cells are trypsinizing, transfer fresh medium to new TC dishes

a) transfer 6, 8, 9, or 9.5 ml and label dishes 1:2.5, 1:5, 1:10, and 1:20, respectively 8) Retrieve cells and verify detachment by visual and microscopic inspection

9) Resuspend cells in 9.5 ml of fresh medium by pipetting up and down a) direct the fluid over the entire plate, avoiding splashing or making bubbles

10) Transfer cell suspension into the dishes prepared in step 7 a) transfer 4.0, 2.0, 1.0, or 0.5 ml into the 1:2.5, 1:5, 1:10 and 1:20 dishes, respectively 11) Seed cells evenly over the TC dish a) take care not to slosh or allow media contact the lids of the dishes

b) gently swirl in both directions, c) gently rock to the four points of the compass

12) Return cells to 37 °C Tissue Culture incubator I. Purify dsRNA (Exp 6) 1) Preheat the elution solution 2) Add the following to the nuclease-digested dsRNA from Tuesday and mix gently with pipettor 10x Binding Buffer 50 µl Nuclease Free Water 150 µl 100% Ethanol 250 µl 3) Add entire 500 µl mixture to a Transcription Reaction Filter Cartridge a) centrifuge 2 minutes on full speed, discard flow through 4) Add 500 µl of Wash Solution to column a) centrifuge 2 minutes on full speed, discard flow through 5) Add 500 µl of Wash Solution to column again

a) centrifuge 2 minutes on full speed, discard flow through b) centrifuge 30 seconds full speed to dry column 6) Transfer the column to a fresh collection tube 7) Add 50 µl preheated Elution solution to column a) centrifuge 2 minutes on full speed 8) Add a second 50 µl preheated Elution solution to column a) centrifuge 2 minutes on full speed 9) Measure concentration of ds RNA a) add 10 µl dsRNA to 90 µl TE and measure A260 RNA concentration in µg/ml = 400xA260 II. Cleave dsRNA into siRNA (Exp 6) 1) Make cleavage reaction mixture

a) calculate the volume of dsRNA that contains 15 µg and the volume of water required c) combine the following reagents in a microcentrifuge tube: dsRNA q.s.* 15 µg (*q.s. = “quantity sufficient”) RNAse III 15 µl 10x RNAse III buffer 5 µl Nuclease Free Water q.s* 50 µl d) Mix gently with pipettor

2) Incubate 1 hour at 37 °C 3) Store remainder of purified dsRNA at –20 °C


IV. Purify siRNA (Exp 6) 1) Add the following to the RNAse III-digested siRNA from part II and mix gently with pipettor 10x Binding Buffer 50 µl Nuclease Free Water 150 µl 100% Ethanol 250 µl 2) Add entire 500 µl mixture to a purification cartridge a) insert cartridge into fresh collection tube centrifuge 2 minutes on full speed, a) discard flow through 3) Add 500 µl of Wash Solution to column a) centrifuge 2 minutes on full speed, discard flow through 4) Add 500 µl of Wash Solution to column again

a) centrifuge 2 minutes on full speed, discard flow through b) centrifuge 30 seconds full speed to dry column 5) Transfer the column to a fresh collection tube 6) Add 50 µl preheated Elution solution to column a) centrifuge 2 minutes on full speed 7) Add a second 50 µl preheated Elution solution to column a) centrifuge 2 minutes on full speed 8) Measure concentration of siRNA a) add 10 µl siRNA to 90 µl TE b) measure A260; RNA concentration in µg/ml = 400xA260 5) Store remaining siRNA at 4 °C


Thursday, May 6 Today you will have a relatively light workload, in compensation for the fact that you will be required to come in to passage cells on Monday, May 10. Today you will passage cells so that they will be ready for you on Monday. You will also learn to count cells and prepare plates at a specific cell density, a skill that will be required for the siRNA transfections you will do in two weeks. Workflow: A. Prepare Medium for Tissue Culture B. Coat Coverslips I. Steps 1-4 C. Examine HeLa Cells II. Steps 1-3 D. Passage HeLa Cells

III. Steps 1-8 E. Count and Plate Cells IV. Steps 1-5 F. Coat Coverslips II. Steps 5-8 I. Coat Coverslips for Use in Transfections (Exp 5)

(see “Protocol #7: “Coating Coverslips With Polylysine” on page 66 for detailed protocol) You will ultimately grow cells on these coverslips, so use sterile technique at all times. 1) Place six SMALL (~1 µl) drops of PBS on the surface of a sterile 10 cm dish

a) one for each coverslip to be coated 2) Place a coverslip on top of each drop a) each student should do three coverslips b) six coverslips total for each group 3) Pipette 50-100 µl of polylysine onto each coverslip 4) Incubate for 1 hr at room temperature 5) Remove the polylysine using a sterile Berol pipette

a) discard in the liquid waste 6) Wash the coverslips with 10 ml sterile PBS a) swirl gently, maintaining orientation (coated side facing up) b) remove PBS using a sterile Berol pipette and discard in the liquid waste 7) Repeat this wash three times 8) Leave the final wash in the dish 9) Store coverslips in sterile PBS in the hood II. Examine HeLa GFP-Cetn2 Cells (Exp 5, 6) Inspect your cells to see which cultures are healthy, and make notations in your lab notebook. Does the 1:2.5 plate you prepared on Tuesday look crowded today? Does the 1:5 or the 1:10 plate look ready to be passaged today? Which did you expect to be ready to be passaged today? III. Passage HeLa GFP-Cetn2 Cells (Exp 5)

If necessary, consult the rules for passaging HeLa cells outlined on page 23. Choose the plate that should be ready, and verify that it is healthy and ready to use. Passage that plate at a density appropriate to ensure you have a plate ready to be passaged on Monday, May 10. What ratio is this? If things don’t match your expectations, it is better to plate a higher volume from a dish at a lower density than to use cells that have overgrown.

NOTE: Transfer the remaining cell suspension to a sterile 15 ml conical tube for use in cell counting


IV. Count Cells 1) Gently mix the cell suspension left over from passaging your cells a) invert the tube several times to resuspend cells that have settled out of suspension 2) Pipet 10 µl of cell suspension into each end of a hemacytometer 3) Place hemacytometer onto tissue culture microscope 4) Count the number of cells in four corners and the middle of each side a) use the “half-on” rule for counting cells that fall on a line - only count those cells for which at least half of the cell is inside the grid 5) Record the number of cells for each of the ten squares counted 6) If the sum of first five squares is roughly equal to the sum of the second five squares

a) the total number of cells counted is the number of cells per µl (make sure you understand why this is true)

8) If the sum of first five squares and the sum of the second five squares are very different - resuspend cells and recount until your numbers are consistent

10) Calculate the volume of cell suspension required to produce the following number of cells:

100 200 500 1,000 2,000 5,000

11) Transfer 2.0 ml fresh medium to each well of a 6-well dish 12) Add the appropriate volumes of cell suspension so that each well has a different number of cells:

well cells 1 100 cells 2 200 cells 3 500 cells 4 1,000 cells 5 2,000 cells 6 5,000 cells


Monday, May 10 You will need to plate cells for two transfections that you will perform Tuesday May 11, DsRed alone and your DsRed-tagged candidate centrosome protein. Although what you need to do today is not difficult, the passaging is more complex compared to the passages you have done to this point, because you will need to plate cells into smaller dishes than those you have used to maintain your cultures. Because you want to maintaining roughly the same cell density you have used previously, you will have to plate fewer total cells into the smaller dish. You will of course also need to passage cells to maintain your stocks for future experiments. Workflow: A. Place Medium and Trypsin in the Water Bath as You Come In B. Transfer Polylysine Coated Coverslips to Six Well Dishes C. Wash Cells with PBS and Passage as Described Below I. While your medium is warming, transfer your coated coverslips to your six well dishes. 1) Transfer three coverslips into each of two wells of your six well dish (see diagram below) a) use sterile technique

b) be sure to keep the coverslips in the same orientation, i.e. coated side facing up NOTE: It’s OK if the PBS on the coverslips dries out before you plate the cells II. Passage Your Cells (Exp 5a, 6) 1) wash, trypsinize, and resuspend cells in 9.5 ml medium (10 ml final volume) as normal 2) Prepare two 10 cm dishes, one each at 1:2.5 and 1:10 for future experiments (e.g. Exp 6) a) add 6.0 ml medium and 4.0 ml of cells to a 10 cm dish b) add 9.0 ml medium and 1.0 ml of cells to a second 10 cm dish NOTE: it is important to put the medium into the wells before the cells. 3) Prepare two wells of a 6-well dish at 1:10 (Exp 5)

a) add 2.0 ml of standard growth medium to the two wells with coverslips b) add 0.16 ml of cells to each well c) you will transfect one of these wells with DsRed, and one with your expression construct

4) Put your 10 cm stock dishes and the 6 well dish in the 37 °C incubator Diagram for plating cells into six well plates for Monday, May 10.


Tuesday, May 11 Today you will transfect the cells you plated yesterday with the DsRed expression construct you created and DsRed alone as a control, following the basic protocol you have used previously. In addition, you will test how good you were at counting cells by fixing, staining, and counting the cells that you plated at specific cell densities on Thursday, May 6 Workflow: A. Prepare Medium for Tissue Culture (if necessary) B. Transfect HeLa GFP-Cetn2 cells I. Steps 1-6 C. Passage Cells (if necessary) D. Fix and Stain Cells for Colony Counting II. Steps 1-8 Experimental Protocols: I. Transfect HeLa GFP-Cetn2 cells (Exp 5a) (see Protocol #4: “Effectine Transfection of Plasmid DNA” on page 63 for detailed protocol) 1) Sterilize your bench tops 2) Combine Transfection Reagents IN ORDER, mix by vortexing for 2 seconds, and incubate 5 minutes

97 µl Buffer EC 4 µl Plasmid DNA (~400 ng) 3.2 µl Enhancer solution

b) Prepare two separate transfection mixes for pEXP-DsRed and pEXP-DsRed-GOI c) Store remaining Effectine, Buffer EC, and Enhancer at 4 °C for later use

3) Add 10 µl Effectene Reagent, mix by vortexing 10 seconds, and incubate 10 minutes 4) During the 10 minute incubation of step 3:

a) Remove medium and rinse each well once with 2.0 ml sterile PBS b) Add 1.5 ml fresh medium to each well

5) Add 0.5 ml medium to transfection components, mix, and transfer dropwise onto cells 6) Return cells to the incubator II. Fix and Stain Cells Plated at Limiting Dilution 1) Place dishes to be stained on ice 2) Wash cells twice with ice-cold PBS 3) Fix cells with ice-cold 95% ethanol for 10 minutes on ice 4) Remove ethanol with a pipet and remove dishes from ice - discard ethanol as liquid waste 5) Add 1.0 ml crystal violet staining solution and incubate 10 minutes at room temperature - make sure that the entire surface of the plate is covered 6) Remove and Recycle crystal violet staining solution 7) Rinse plates with distilled water a) add 10 ml water to each dish b) swirl gently and decant rinse c) repeat until no more stain comes off plates 8) Leave plates to dry overnight at room temperature


Thursday, May 13 Today you will harvest the coverslips from the transfections you did on Tuesday and process them for IIF. A table listing which coverslips are to be handled in what manner, and a diagram that should help you keep track of the various manipulations you need to do are found following the detailed experimental protocols. Workflow: A. Prepare Medium for Tissue Culture B. Harvest Coverslips From 6-Well Plate I. Steps 1-3 C. Perform IIF II. Steps 1-6 D. Coat Coverslips III. Steps 1-8 E. Passage Cells Experimental Protocols: I. Harvest Coverslips (Exp 5a)

(see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol) 1) Transfer coverslips from 6 well plate into a 24 well dish - consult the diagram on page 49 to help you keep track of the manipulations 2) Add 0.15 ml F/T to each well and incubate 10 minutes at room temperature 3) Wash coverslips five times with ~0.5 ml non-sterile PBS


II. Indirect Immunofluorescence (IIF) (Exp 5a) (see Protocol #3 “Indirect Immunofluorescence (IIF) Analysis” on page 62 for detailed protocol) 1) Add 0.25 ml of IIF Blocking buffer, incubate 60 minutes at room temperature in the dark* 2) Remove blocking buffer, add 0.15 ml antibody mixes as follows, and incubate 60 minutes in the dark a) consult the diagram on page 49 to help you keep track of the manipulations - coverslip #1, 4: no antibody - coverslip #2, 5: mouse anti-αTubulin - coverslip #3, 6 mouse anti-γTubulin 3) Remove 1° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 4) Remove final PBS wash, add 0.15 ml secondary antibody, and incubate 60 minutes in the dark 5) Remove 2° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol) III. Coat Coverslips for Use in Transfections (Exp 6)

(see “Protocol #7: “Coating Coverslips With Polylysine” on page 66 for detailed protocol) 1) Following the same procedure used last week, each student should coat six coverslips

a) for a total of 12 coverslips for each group 2) Wash the coverslips four times with 10 ml sterile PBS 3) Leave the final wash in the dish, and store coverslips in the hood IV. Passage Cells (Exp 5b, 6)

Choose a plate of HeLa GFP-Cetn2 cells that is healthy and ready to be passaged. Passage that plate at a density appropriate to ensure you have a plate ready to be passaged on Monday, May 17. What ratio is this? If necessary, consult the rules for passaging HeLa cells outlined on page 23.


Diagram for harvesting cells on Thursday, May 13:


Monday, May 17 You will need to plate cells for two transfections that you will perform Tuesday May 18. What you need to do today is more complex than what you have done to this point for several reasons; you will need to plate cells at two different cell densities for two different types of transfections (expression constructs vs siRNA), and you will need to count cells in order to plate them at a specific cell density for siRNA transfection, for which cell density is critical to success. You will of course also need to passage cells to maintain stocks in case you need to repeat any experiments. So please pay close attention to the instructions and clearly mark your plates so that you know which cells to use for tomorrow’s experiments. For the subsequent experiments, it is absolutely critical that your cells are healthy and ready to passage. If your cells are not useable for the passages described below, please get cells from the TAs. Workflow: A. Place Medium and Trypsin in the Water Bath as You Come In B. Transfer Polylysine Coated Coverslips to Six Well Dishes C. Wash Cells with PBS and Passage as Described Below I. While your medium is warming, transfer your coated coverslips to your six well dishes. 1) Transfer three coverslips into each of four wells of your six well dish (see diagram below) a) use sterile technique, and maintain orientation (i.e. coated side facing up) NOTE: It’s OK if the PBS on the coverslips dries out before you plate the cells II. Passage Your Cells (Exp 5b, 6) 1) Wash, trypsinize, and resuspend cells in 9.5 ml medium (10 ml final volume) as normal 2) Prepare one 10 cm dish at 1:2.5 (6.0 ml med., 4.0 ml cells), and one at 1:10 (9.0 ml med., 1.0 ml cells) 3) Add 2.0 ml of standard growth medium to all six wells of the 6-well dish 4) Using a hemacytometer, determine the concentration of cells in your suspension a) calculate the volume of this cell suspension required to obtain 5x103 cells 5) Prepare two wells of the 6-well dish at 1:10

a) add 0.16 ml of cells to wells 1 and 4 (see diagram below) 6) Prepare four wells of the 6-well dish at 5x103 cells per well b) add 5x103 cells to the remaining four wells (e.g. wells 2, 3, 5, and 6) d) gently rock the dish to distribute cells evenly 5) Put your 10 cm stock dishes and the 6 well dish in the 37 °C incubator Diagram for plating cells into six well plates for Monday, May 17.


Tuesday, May 18 Today you will use the cells you plated yesterday in two different transfection techniques. You will use Effectene reagent to transfect the cells you passaged at 1:10 with DsRed and your expression construct, just as you did last week. You will use a different reagent, Oligofectamine, to transfect the remaining cells with siRNAs. The protocol for Oligofectamine is similar to the Effectene protocol, but the differences are critical. Therefore please pay close attention to the specific instructions for each transfection. Note: It is very important to maintain proper sterile technique, today

- you will be using antibiotic free medium for the siRNA transfections Workflow: A. Place Medium and Trypsin in Water Bath as You Come In B. Transfect 1:10 Wells (Wells #1 and 4) With DsRed or your Expression Construct C. Transfect Four 5x103 Wells (Wells #2, 3, 5, and 6) With siRNA Experimental Protocols: I. Transfect wells #1 and #4 with DsRed Expression Constructs, and Arrest in S-phase (Exp 5b) (see Protocol #4: “Effectine Transfection of Plasmid DNA” on page 63 for detailed protocol) 1) Sterilize your bench tops 2) Prepare two transfection mixes by mixing the following reagents IN ORDER as follows:

97 µl Buffer EC 97 µl Buffer EC 4 µl pEXP-DsRed (~400 ng) 4 µl pEXP-DsRed-GOI (~400 ng) 3.2 µl Enhancer solution 3.2 µl Enhancer solution

a) mix by vortexing for 2 seconds, and incubate 5 minutes at room temp b) Store remaining Effectine, Buffer EC, and Enhancer at 4 °C for later use

3) Add 10 µl Effectene Reagent, mix by vortexing 10 seconds, and incubate 10 minutes at room temp 4) During the 10 minute incubation of step 3:

a) Remove medium from wells 1 and 4 b) Rinse wells 1 and 4 once with 2.0 ml sterile PBS b) Add 1.5 ml fresh medium to wells 1 and 4

5) Add 0.5 ml medium to transfection components, and mix a) transfer pEXP-DsRed-GOI transformation mix dropwise into well 1 b) transfer pEXP-DsRed (control) transformation mix dropwise into well 4

6) Return cells to the incubator II. Transfect wells # 2, 3, 5, and 6 with siRNA (Exp 6) 1) Sterilize your bench tops 2) Calculate the µM concentration of your siRNA pool using last week’s µg/ml calculation a) concentration in µM = concentration in µg/ml ÷ 9 b) see siRNA manual for explanation 3) Make a 20 µM solution of your siRNA pool a) if your siRNA is greater than 20 µM, use SFDME to make 30 µl of a 20 µM solution

(SFDME = Serum and antibiotic Free DME) b) if your siRNA is below 20 µM, note the concentration and use 25 µl in step 5 below


4) Dilute Oligofectamine a) mix the following reagents in a microcentrifuge tube 30 µl Oligofectamine 195 µl SFDME b) mix by vortexing 5 seconds c) incubate 5 minutes at room temperature 5) Dilute siRNAs a) prepare two separate tubes, one for your GOI siRNA and one for the control siRNA b) for each siRNA, mix the following reagents: 25 µl of 20 µM siRNA (your pool or control) 500 µl SFDME NOTE: If your siRNA is below 20 µM, note the concentration and use 25 µl 6) Add 90 µl Oligofectamine mix to each siRNA dilution a) mix by vortexing 5 seconds b) incubate 15 minutes at room temperature 7) Wash wells #2, 3, 5, and 6 with PBS (do this during the 15 min siRNA/Oligofectamine incubation)

a) Remove medium and rinse wells #2, 3, 5, and 6 once with 2.0 ml sterile PBS b) Add 0.75 ml fresh SFDME to wells #2, 3, 5, and 6 NOTE: Add SFDME only DO NOT ADD STANDARD GROWTH MEDIUM!

8) Transfer 250 µl of each transfection mix dropwise to wells #2, 3, 5, and 6 a) gently mix reagents prior to transfer b) add siRNA-GOI mix to wells 2 and 3 c) add control mix to wells 5 and 6 9) Incubate 4 hour at 37 °C 10) Add 500 µl growth medium + 30% FBS to all four 1:20 wells (TAs will do this)


Thursday, May 20 Today you will harvest the cells from the transfections you did on Tuesday, May 18. First, you will harvest the coverslips from both transfections and process them for IIF. However, you will also harvest the remaining cells for immunoblot analysis (which you will do next week). For this procedure, it is important to avoid foaming the medium or making bubbles as much as possible. Luckily, it will no longer be necessary to maintain sterile technique, as the cells will not be returned to the incubator, and it will be easier to handle the six well plate in such a manner as to avoid bubbles. One member of each group should work on the GFP expression construct transfections (Section I below), and the other group member should work on the siRNA transfections (Section II below). Those of you working on the siRNA transfections will have to keep track of two different fixation conditions. Therefore, pay close attention at each step to ensure that you will be able to collect the data that will allow you to determine whether your proteins localize to centrosomes, and whether they have any centrosomal function. A diagram that should help you keep track follows the detailed experimental protocols. Workflow: A. Harvest The Coverslips From Your Six Well Plate I. Steps 1-3 II. Steps 1-3 B. Perform IIF III. Steps 1-2 IV. Steps 1-2 C. Harvest The Remaining Cells From Your siRNA Transfections V. Steps 1-9 E. Perform IIF III. Steps 3-4 IV. Steps 3-4 Experimental Protocols: I. Harvest Coverslips from the DsRed transfections (wells 1 and 4) (Exp 5b)

(see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol) 1) Transfer coverslips from wells 1 and 4 of the 6 well dish into a 24 well dish a) DsRed-GOI transfected coverslips = coverslips #1-#3 b) DsRed (control) transfected coverslips = coverslips #4-#6 - consult the diagram on page 55 to help you keep track of the manipulations 2) Add 0.15 ml F/T to each well and incubate 10 minutes at room temperature 3) Wash coverslips five times with ~0.5 ml non-sterile PBS


II. Harvest coverslips from the siRNA transfections (wells 2 and 5) (Exp 6)

(see Protocol #1 “Fixing Cells on Coverslips” on page 60 for detailed protocol) 1) Transfer coverslips from wells 2 and 4 of the 6 well dish into a 24 well dish a) GOI-specific siRNA transfected coverslips = coverslips #7-#9 b) control siRNA transfected coverslips = coverslips #10-#12 - consult the diagram on page 55 to help you keep track of the manipulations


2) Fix cells with two different fixatives a) Add 0.15 ml FA (4% formaldehyde alone) to coverslips #7 and #10 b) Add 0.15 ml FT (4% Formaldehyde + 0.2% triton) to coverslips #8, 9, 11, and 12

c) incubate 10 minutes at room temperature 3) Wash coverslips five times with ~0.5 ml non-sterile PBS a) avoid cross contamination of FA and FT when removing fixative and first wash

b) discard formaldehyde and first PBS wash into a labeled waste beaker c) discard subsequent washes as standard waste

III. Indirect Immunofluorescence (IIF) analysis of the DsRed transfections (Exp 5b) (see Protocol #3 “Indirect Immunofluorescence (IIF) Analysis” on page 62 for detailed protocol) 1) Add 0.25 ml of IIF Blocking buffer, incubate 60 minutes at room temperature in the dark* 2) Remove blocking buffer, add 0.15 ml antibody mixes as follows, and incubate 60 minutes in the dark a) consult the diagram on page 55 to help you keep track of the manipulations - coverslip #1, 4: no antibody - coverslip #2, 5: mouse anti-αTubulin - coverslip #3, 6 mouse anti-γTubulin 3) Remove 1° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 4) Remove final PBS wash, add 0.15 ml secondary antibody, and incubate 60 minutes in the dark 5) Remove 2° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol) IV. Indirect Immunofluorescence (IIF) analysis of siRNA transfections (Exp 6) (see Protocol #3 “Indirect Immunofluorescence (IIF) Analysis” on page 62 for detailed protocol) 1) Add 0.25 ml of IIF Blocking buffer, incubate 60 minutes at room temperature in the dark* 2) Remove blocking buffer, add 0.15 ml antibody mixes as follows, and incubate 60 minutes in the dark a) consult the diagram on page 55 to help you keep track of the manipulations - coverslip #7, 10: no antibody - coverslip #8, 11: mouse anti-αTubulin - coverslip #9, 12 mouse anti-γTubulin 3) Remove 1° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 4) Remove final PBS wash, add 0.15 ml secondary antibody, and incubate 60 minutes in the dark 5) Remove 2° antibodies, avoiding cross contamination, and wash cells 5 times with non-sterile PBS 6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61 for detailed protocol) V. Harvest the remaining siRNA-transfected cells (wells 3 and 6) (Exp 6) NOTE: It is important to avoid foaming the medium or making bubbles. 1) Transfer 1.0 ml of medium from wells 3 and 6 into separate clean, labeled microcentrifuge tubes a) take care not to cross contaminate your samples 2) Remove and discard the remaining medium from all wells 3) Wash wells 2, 3, 5, and 6 with 2.0 ml PBS a) remove any residual PBS 4) Add 0.1 ml trypsin to wells 2, 3, 5, and 6 a) incubate 2-3 minutes at 37 °C, or as long as it takes to dissociate the cells 5) Gently resuspend the trypsinized cells using the medium you saved in step 1 a) do not cross contaminate between wells

e.g. use the medium from well 3 to harvest the cells in wells 2 and 3 and use the medium from well 6 to harvest the cells in wells 5 and 6


6) Gently pellet the cells in a microfuge at 1000 g for 5 minutes a) using a P1000, remove and discard the supernatant

b) take care not to disturb the very small cell pellet - it is better to leave a small amount of medium than to remove any cells 8) Gently resuspend the cell pellets with 0.25 ml PBS 9) Gently pellet the cells in a microfuge at 1000 g for 5 minutes

a) remove and discard the supernatant b) take care not to disturb the very small cell pellet c) give your cells to the TAs, who will plunge freeze them in liquid nitrogen Diagram for harvesting cells on Thursday, May 20:


Tuesday, May 25 This week you will be using Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques to analyze the siRNA-transfected cells you harvested last week in order to determine whether depletion of your candidate protein had any affect on the levels of Mps1. Today, you will boil your cell pellets in a buffer designed to denature and coat the proteins with SDS, use SDS-PAGE to separate the proteins in your samples by size, and transfer the separated proteins to nitrocellulose filters (be sure to note which gel and location you use for your samples!). Once your samples have been transferred onto nitrocellulose filters (or “blots”), you will label your blots and place them at 4 °C in blocking buffer until Thursday, when you will continue the procedure. During the time that your gels are running and transferring, you will be using the fluorescence microscopes to analyze the IIF you did last week. NOTE: Today you will be boiling samples in microcentrifuge tubes, which can pop open in the process. Therefore, we must insist that you wear safety glasses for this portion of the procedure. Workflow: A. Lyse and Boil Your Cell Pellets B. Load Your Lysates onto SDS-PAGE Gels C. Transfer of Your Samples From SDS-PAGE Gels onto Nitrocellulose D. Examine IIF Slides by Fluorescence Microscopy Experimental Protocols: I. Lyse and Boil Your Cell Pellets (Exp 6) 1) Take your samples from the ice bucket at the front of the room 2) Add 20 µl of 1X SDS Sample Buffer to each sample a) vortex each sample for 60 seconds b) boil samples i) wear safety glasses ii) insert your tubes into a 95 °C heating block iii) incubate at 95 °C for 5 minutes iv) quickly (but carefully!) recap any tube that pops open 3) Spin your samples at full speed for 3 minutes to collect condensed liquid and pellet insoluble material II. Load your lysates onto SDS-PAGE gels (Exp 6) 1) Load your samples onto three adjacent wells of one SDS-PAGE gel using a P20 a) load 10 µl of Molecular Weight Marker in your first well

b) load 20 µl of each sample in the second and third wells c) be sure to note where you loaded your samples i) note which gel (A, B, C, or D) and lanes (1-3, 4-6, or 7-9) your samples are in ii) note the order of your samples on that gel (e.g. A1=MW, A2=siGLO, A3=siCLIC1) 2) The TAs will run the gels at 70 mA constant current for approximately 1 hour


III. Transfer of your samples from SDS-PAGE gels onto nitrocellulose (Exp 6) 1) The TAs will set up the transfer of the gels to nitrocellulose

a) transfer will be carried out at 90 volts for one hour 2) The TAs will take down the transfers, cut the blots, and distribute them 3) Place your blot into a small plastic container a) mark the outside of the container with your initials 4) Using a grease pencil or red correcting pencil, mark a corner of your blot with your initials 5) Stain your blot with the reversible protein stain Ponceau S a) pour enough Ponceau S into the container to cover your blot b) let your blot sit for ~5 minutes c) recycle the stain back into the original bottle or a collection beaker d) rinse the blot several times with dH2O i) NOTE: DO NOT use tap water e) note the relative protein level in each sample i) were your siGLO and siRNA-GOI samples equally loaded? ii) was there more protein in one lane compared to the other? 7) Drain any water and add ~50 ml blocking buffer a) cover your dish with plastic wrap 8) Store your blots in blocking buffer at 4 °C until Thursday


Thursday, May 27 Today you will analyze the immobilized proteins from your siRNA transfections by immunoblotting. The procedure is analogous to the procedure you used for indirect immunofluorescence (IIF) in that you will incubate your blots with a primary antibody, followed by fluorescently labeled secondary antibodies. The secondary antibodies we will use are labeled with infrared dyes that will be excited using a machine called the Odyssey scanner that uses a laser to stimulate the dyes and a microscope to detect the infrared emissions. The machine has two different lasers and can detect the emissions from two different dyes, so just like IIF we can image two different secondary antibodies at once. However, because SDS-PAGE separates proteins by size, immunoblot analysis has the additional advantage that you can look at two proteins at the same time with a single secondary antibody, provided that the proteins have a different molecular weight. You will stain your blots with an antibody to α-Tubulin (to estimate the relative amount of protein in each lane), and with an antibody against Mps1 to determine if disrupting your candidate protein had any effect on Mps1 degradation. During the time that your blots are incubating in primary and secondary antibodies, you will be using the fluorescence microscopes to continue analyzing the IIF you did last week. Workflow: A. Incubate Your Blots in Primary Antibody Mix Steps 1-2 B. Wash Your Blots Step 3 C. Incubate Your Blots in Secondary Antibody Mix Step 4 D. Wash Your Blots Step 5 E. Scan Your Blots on the LI-COR Odyssey Step 6 F. Examine IIF Slides by Fluorescence Microscopy I. Immunoblot Analysis (Exp 6) 1) Remove your blots from 4 °C 2) Add primary antibody to your blot a) discard blocking buffer b) add 10 ml of primary antibody mixture to your container:

mouse anti-αTubulin + rabbit anti-Mps1 c) re-cover your container with plastic wrap d) incubate with shaking for 60 minutes at room temperature 3) Wash your blots a) discard the primary antibody solution b) add 20 ml PBST to your container c) incubate with shaking for 5 minutes at room temperature d) discard the PBST and wash twice more 4) Add secondary antibody to your blot a) discard the final PBST wash b) add 10 ml of secondary antibody mix to your container i) secondary antibody mix contains IRDye800 anti-rabbit and Alexa690 anti-mouse c) cover your container with aluminum foil and label with your initials d) incubate with shaking for 60 minutes at room temperature


5) Wash your blots a) discard the secondary antibody solution b) add 20 ml PBST to your container c) incubate with shaking for 5 minutes at room temperature d) discard the PBST and wash twice more e) leave the blot in the final PBST wash (i.e. do not discard the final wash) 6) Scan your blots a) two groups at a time can take their blots to the Odyssey scanner on the 2nd floor b) rinse the scanning bed with water c) place your blot, protein side down on the scanning bed d) follow the TA’s instructions to scan your blots


Protocols

Below are those protocols that will be most commonly used throughout the course. In the daily workflows these protocols are presented in an abbreviated form. Below are presented detailed versions of protocols for fixing cells, mounting coverslips, performing IIF, transfecting cells, transforming bacteria, isolating plasmid DNA, and coating coverslips. Protocol #1: Fixing Cells on Coverslips 1) Transfer coverslips into a 24 well plate

a) use jeweler’s forceps to transfer coverslips into a 24 well plate - one coverslip per well from your dish - maintain orientation so that cells continue to face the ceiling

2) Fix cells a) add 0.15 ml appropriate fixative (see below) to each well - wear gloves, formaldehyde is toxic! b) incubate 10 minutes at room temperature in the dark*

3) Wash out fixative a) remove formaldehyde solution with a non-sterile† Berol pipette - discard formaldehyde into a labeled waste beaker b) add ~0.5 ml non-sterile PBS† to each well - discard this first PBS wash with the formaldehyde waste c) wash coverslips 4 more times with ~0.5 ml PBS

- use the same pipette for all washes - subsequent washes can be discarded as standard waste

Fixative Solutions: F: 4% Formaldehyde in PBS F/T: 4% Formaldehyde + 0.2% Triton X-100 in PBS * “In the dark” is achieved by placing dish into a cubby under your bench † to avoid unnecessary expense, please use non-sterile PBS and pipettes for IIF

do not use the same sterile PBS that you use for cell culture


Protocol #2: Mounting coverslips 1) Prepare slides

a) label the frosted end of a slide with initials and date b) place drops of mounting medium in a line on a slide

- four per slide is recommended - up to six per slide is possible

2) Transfer coverslips onto slides

a) invert each coverslip onto a drop of mounting medium b) keep track of coverslips - default, coverslip #1 closest to frosting, coverslip #4 is farthest from frosting

3) Remove excess mounting medium

a) GENTLY press coverslips with forceps to remove excess mounting medium b) CAREFULLY dab with a dry paper towel to blot excess mounting medium c) CAREFULLY dab with a damp paper towel to clean coverslip

4) Seal coverslips

a) once coverslips are dry, seal around edges of coverslips with nail polish


Protocol #3: Indirect Immunofluorescence (IIF) Analysis Transfer coverslips to a 24 well plate and fix cells (see Protocol #1 “Fixing Cells on Coverslips” on page 60) 1) Block non-specific protein binding sites a) remove and discard the final PBS wash b) add 0.25 ml of IIF Blocking buffer c) incubate 60 minutes at room temperature in the dark* 2) Incubate with primary antibodies a) remove blocking buffer and add 0.15 ml of appropriate primary antibody mix b) incubate 60 minutes at room temperature in the dark* 3) Wash out primary antibodies

a) remove and discard primary antibody solutions - use a pipettor and change tips to avoid cross contamination - antibodies can be discarded as standard waste b) wash cells 5 times with ~0.5 ml PBS - washes can be discarded as standard waste

4) Incubate with secondary antibodies

a) remove final PBS wash b) add 0.15 ml of the appropriate secondary antibody with or without Hoechst

c) incubate 60 minutes at room temperature in the dark* 5) Wash out secondary antibodies a) remove and discard secondary antibody solutions

- use a pipettor and change tips to avoid cross contamination - antibodies can be discarded as standard waste b) wash cells 5 times with ~0.5 ml PBS - washes can be discarded as standard waste - do not remove final wash

6) Mount coverslips (see Protocol #2 “Mounting Coverslips” on page 61) * “In the dark” is achieved by placing dish into a cubby under your bench † use non-sterile PBS for IIF, do not use the same sterile PBS you use for culture


Protocol #4: Effectine Transfection of Plasmid DNA 1) Sterilize your bench tops a) don gloves and wipe with 70% ethanol b) wipe down bench top with disinfectant c) wipe down bench top with 70% ethanol 2) Combine Transfection Reagents: a) Add the following IN ORDER to a microcentrifuge tube

(volumes determined by size of dish using the following table)

Buffer EC (calculated based on volume of DNA [VEC=VFinal-(VDNA+VEn]) Plasmid DNA (calculated based on DNA concentration) Enhancer solution (constant based on growth area, see table)

Dish Size DNA (Step 2)

Final volume (Step 2)

Enhancer (Step 2)

Effectine (Step 4)

Volume to Dish (Step 5)

Volume to Mix (Step 6)

24-well 0.2 µg 60 µL 1.6 µL 5 µL (1)† 0.25 0.35 6-well 0.4 µg 100 µL 3.2 µL 10 µL (2) 1.5 0.6 60 mm 1.0 µg 150 µL 8.0 µL 25 µL (5) 3.0 1.0

3) Mix Transfection Reagents

a) vortex for 2 seconds c) Incubate 5 minutes at room temperature d) Store remaining Effectine, Buffer EC, and Enhancer at 4 °C for later use

4) Make transfection complexes

a) add Effectene Reagent (volume based on amount of Enhancer used, see table) b) Mix by vortexing 10 seconds c) Incubate 10 minutes at room temperature

5) Wash cells (during the 10 min incubation of step 3) a) follow sterile technique practices b) remove medium from cells and discard in waste beaker - be careful not to generate bubbles when removing the medium c) rinse cells on dish surface once with sterile PBS

add the PBS slowly and gently, being careful not to disturb any coverslips use 0.5 ml PBS for 24 well dish use 2.0 ml PBS for 6 well dish use 4.0 ml PBS for 60 mm dish remove PBS from dish and discard in waste beaker d) Add fresh medium to cells be careful not to generate bubbles when adding the fresh medium add 0.25 ml medium to the wells of a 24 well dish use 1.5 ml medium to the wells of a 6 well dish use 3.0 ml medium to 60 mm dishes

6) Add transfection mix to cells a) add 1.0 ml fresh medium to transfection complexes created in step 3 b) mix with pipettor be careful not to generate bubbles be careful not to draw medium into the barrel of the pipettor c) transfer transfection mixture dropwise onto cells

7) Return cells to the incubator


Protocol #5: B. Bacterial Transformation - TAs will thaw competent DH5α cells on ice, and dispense them into individual tubes - each group should take a tube (or tubes) and place them IMMEDIATELY on ice - it is important to keep the cells as cold as possible until the heat shock in step 2 Label your tube(s) with your initials and a description of the sample (e.g. “+” and “-“) 1) Add DNA

a. pipet 1 µl of the appropriate DNA mix into individual tubes of DH5α cells b. gently mix sample and cells c. incubate 10 minutes on ice

2) Heat shock a. place tubes containing the mixture of cells and DNA in the 42 °C water bath b. incubate exactly 45 seconds c. return tubes to ice for 1 minute

3) Recover a. add 250 µl SOC medium to each tube - use sterile technique, SOC is extremely rich medium b. incubate cells with shaking for 60 minutes at 37 °C

4) Plate cells a. spot 50 µl from each tube onto a separate antibiotic containing plate NOTE: for less efficient transformations, plate more of the transformation mixture b. spread the cells evenly using a sterile spreader to distribute the liquid

c. incubate plates in 37 °C bacterial incubator overnight Note:

NEVER incubate bacterial cells in the same incubator with tissue culture cells! NEVER handle tissue culture cells immediately after working with bacteria! Wash hands with soap and water first.


Protocol #6: Plasmid “Miniprep” DNA Isolation 1) Retrieve your bacterial cultures from 4° C a) resuspend bacteria by vortexing 2) Each group member will perform three “minipreps”

a) each group will perform a total of six minipreps 3) Using a P1000 pipettor, transfer 1.5 ml of culture to a microcentrifuge tube a) avoid aspirating liquid into pipette barrel b) transfer 0.75 ml twice using the same pipette tip c) change pipette tips between cultures 4) Pellet bacteria a) spin in microcentrifuge at half speed for 2 minutes 5) Remove and discard supernatant into a waste beaker 6) Resuspend bacterial pellet

a) add 250 µl Buffer P1 +RNAse A b) respuspend by vortexing

7) Lyse bacteria a) add 250 µl Buffer P2

b) mix by gently inverting 2-3 times c) do not vortex d) incubate no longer than 5 minutes at room temperature 8) Neutralize and precipitate genomic DNA and cell debris

a) by adding 350 µl Buffer N3 b) mix by inverting 5-6 times c) do not vortex 9) Pellet genomic DNA and cell debris

a) spin samples in microcentrifuge at full speed for 10 minutes 10) Bind plasmid DNA onto purification column a) place a column into a collection tube

b) pour supernatant onto the column c) spin columns in microcentrifuge at full speed for 1 minute d) discard supernatant 11) Wash plasmid DNA a) add 0.75 ml Buffer PE to column b) spin columns in microcentrifuge at full speed for 1 minute d) discard supernatant 12) Dry column a) return column to collection tube and spin 1 minute 13) Elute plasmid DNA a) place columns in microcentrifuge tubes b) add 50 µl Buffer EB directly onto column bed c) DO NOT TOUCH COLUMN BED WITH PIPET TIP! d) spin columns in microcentrifuge at full speed for 1 minute e) discard column NOTE: In this step the plasmid DNA is released from the column into the elution buffer and collected by

centrifugation. This collected plasmid DNA is referred to as a “miniprep.”


Protocol #7: Coating Coverslips With Polylysine You will ultimately grow cells on these coverslips, so use sterile technique at all times. 1) Place a SMALL (~1 µl) drops of PBS on the surface of a 10 cm dish for each coverslip to be coated space drops at least 2 cm apart space drops at least 1 cm from the side of the dish

NOTE: this spacing of coverslips is critical! (see below) 2) Place a coverslip on top of each drop of PBS

NOTE: it is critical coverslips do not touch each other or the side of culture dish! - this will allow polylysine to contact the surface of the dish (see below) 3) Pipette 50-100 µl of polylysine onto each coverslip

NOTE: it is critical that polylysine does not come into contact with the dish! - this will allow polylysine to be drawn under the coverslip by capillary action

- this will in turn cause the coverslips to float, and will result in coating of both sides (both are bad!)

4) Incubate for 1 hr at room temperature 5) Remove the polylysine using a Berol pipette discard polylysine in the liquid waste 6) Wash the coverslips with 10 ml sterile PBS pipette 10 ml PBS onto coverslips swirl gently, maintaining orientation (coated side facing up) remove PBS using a Berol pipette and discard in the liquid waste 7) Repeat this wash three times 8) Leave the final wash in the dish

Store coverslips in PBS

Spring 2010 Eukaryotic Cell and Developmental … Dossier...MG 602, Spring 2010 Lab Manual page 3 MG 602 Spring 2010 Eukaryotic Cell and Developmental Biology Laboratory Lab Manual

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