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EBBEP Workshop FP Plasmid Digest Lab
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EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Mar 26, 2015

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Page 1: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

EBBEP Workshop

FP Plasmid Digest Lab

Page 2: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Where the FP came from

There are 6 different FP used for this lab available through EBBEP

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Of the 6 proteins; 3 are from the Aequorea victoria jellyfish and 3 are from Discosoma striata anemone

Discosoma striata anemone Aiquorea victoria jelly

Page 3: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Structure of FP proteins

All of the proteins used by EBBEP have a similar structure.They are all monomer barrel shaped molecules with a central chromatophore

Small changes in the Amino acids in or directlyAround the chromatophore cause changes in color

Page 4: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

How were the proteins made?All proteins were made by Mutagenesis of

the natural occurring proteins found in types of Cnidarians.

Proteins made from Jellyfish GFPEmerald (Green)Venus (yellow)Blue

Proteins made from anemone RFPTangerine (orange/pink)Cherry (Dark pink)Grape (purple)

Page 5: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Using evolution

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Since the proteins come from two different “roots” of evolution you can see the inheritance in the sequences of the proteins

Page 6: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

The FP plasmids

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Made from pRSET vector

FP gene is less than 730bp

Only difference in plasmids is the FP gene

Page 7: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Restriction enzymes

Type 2 restriction endonucleases are used in this lab. (most common)

Cut DNA at a specific sequence.Enzymes used

HindIII

AhdI

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Page 8: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Why are there restriction enzymes?

Evolved by bacteria to protect against viral infection

Over 3000 known enzymes

Page 9: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Challenges in making the labNeeded an enzyme that cut green based genes different than red based genes. That’s easy! Several to choose from

Finding enzymes that cut the plasmid into a reasonable number of bands and have bands of an easily visible size on a gel Not so easy

Page 10: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Plasmid maps

Provided are 2 maps (red based and green based)

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Page 11: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Reading the maps and predicting size

HindIII only cuts the plasmid once making 1 linier piece the full size of the plasmid ~3623bp

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Page 12: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Reading the maps and predicting size

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AhdI cuts 2 times producing 2 fragments

Size of fragment 12555-445= 2100bp

Size of fragment 2 has 2 way to calculate

3623-2110 = 1513bp Or3623-2555+445= 1513

Page 13: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Teacher set-up tips

Get labeling tubes early!DNA, Buffers, Water, loading dye, and marker/ ladder are directly aliquotted from stocks supplied

Can be completed a few days in advance lf you have a freezer. More if you have a frosted freezer

Page 14: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Teacher Set-Up tips

Enzymes must be diluted.Receive a stock enzyme tube and a dilution tube for each enzyme.

Add contents of one tube to the other (pre measured for you) Must be done no earlier than 24 hrs before or enzymes will not work as well

Page 15: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Teacher set up tips

Sterile 1.5 snap cap tubes and filter pipette tips are ideal.

Will work without the Sterile but you will possibly see fuzzier bands in the gel.

Cap tubes before you give them out to help decrease the contamination

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Page 16: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Teacher Set-Up tips

Make sure you plan for a long day. Staining gels with EtBr takes time.

If you don’t have a digital imaging system then remember you digital camera. QuickTime™ and a

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Page 17: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Things to stress with students:Set up Digests on ICE!Concentrations are important (More is not better)

Add reagents in Correct order. Water first ,Buffer, DNA, Enzymes are always last.Reason for Enzymes being last: Enzymes are sensitive to conditions outside of their normal range. Strong buffer solutions can effect the efficiency of the digest

Page 18: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Lab Procedure

Each group of 2 students sets up 3 tubes. Negative control (no enzyme)

An undigested plasmid shows as usually 2 or more bands (nicked/open circle, super super coiled,multimers

TravelsFaster

TravelsSlower

bs.kaist.ac.kr

Page 19: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Lab procedure

HindIII digest-Should make 1 linier fragment. Shows true size of plasmid

AhdI digest- determines if plasmids are from red (2 bands) or green (1band) line.

2 Groups per gel with marker in middle.Best to give 1 green and 1 red to each gel.

Page 20: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Lab procedure

Incubate at 37C.Freeze over night (fridge will work for 24hrs)

Run gel. The bands are far enough apart you can run it pretty fast… if you can live with the smile bands on the gel

Page 21: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Keep it clean and don’t digest too longContaminations of DNases can be devastating. Lane 8 has little to none

DNase activityLane 1 has a large amount of DNase activity

biosyn.com

Page 22: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Things that can go wrong

Impeded digestion due to incorrect set up. (too much or too little buffer etc.)

Star digest activityUnder non-standard reaction conditions, some restriction enzymes are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered specificity has been termed star activity"

Page 23: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Star digestion example

Examples:EcoRI is

supposed to only cut GAATTC but, under extreme conditions, it might possibly cut CAATTC also.

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http://www.fermentas.com/en/support/technical-reference/restriction-enzymes/star-activity

Page 24: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Star digests

Things that can cause Star digestsToo much glycerol in reaction. (More is not better)

Incorrect buffer of buffer concentration

Extended digest times. Don’t leave them over night

Page 25: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Expected results

Plasmids made from the Green FP (jellyfish) digest into single bands for both HindIII and AhdI

Plasmids made from the Red FP (anemone) digest into a single band for HindIII and 2 bands for AhdI

Page 26: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Nicked circle

1 2 3 4 5 6 7 8

Undigested green

base

d plasm

id

Undigested red based pla

smid

Gre

en Hind

III

Red H

indIII

Gre

en AhdI

Red A

hdI

Marker

Supercoiled

Multimers

2 distinct bands showing red protein origins

Sample gel from small FP plasmid digest lab

Lin

ier

Page 27: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Making a standard curve

Measure to leading edge of each band in the marker/ ladder

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Page 28: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Standard Curve

Graph of lambda HindIII marker (base pair vs. Distance migrated)

Using semi log graph paper

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Page 29: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Using graph to find size of Unknowns

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Page 30: EBBEP Workshop FP Plasmid Digest Lab. Where the FP came from There are 6 different FP used for this lab available through EBBEP Of the 6 proteins; 3 are.

Standard curves

http://www.phschool.com/science/biology_place/labbench/lab6/standcur.html