Molecular Biology II Lecture 1 OrR. Restriction Endonuclease (sticky end)

Molecular Biology IILecture 1

OrR

Restriction Endonuclease (sticky end)

Restriction Endonuclease (blunt end)

Restriction Endonuclease Type II

Restriction Endonuclease (Type IIS)

Neoschizomers

Ligation (sticky end)

Ligation (Blunt end)

Problem

• I know a organism which contain a gene X • Partial sequence of the gene is known to me.• I want to isolate the gene (only coding region

or control region with the coding region from the organism. How will I do it?

PCR

Primer?

Plasmid

How we insert DNA fragment in plasmid????

Adapter In Primer

• What is it?• Function?• Which side of the primer is it added?• If it is not complementary to the sequence

how the PCR happen (home work). Figure required!!!!!

How we know if my plasmid transformed?

Selectable marker

Other cloning vectorTable 1.1: Selected Cloning Vectors and Maximum Insert Sizes____________________________________________________________Vector Maximum Insert Size____________________________________________________________Plasmids 15 kbPhagemids 15 kbPhage lambda 23 kbCosmids 44 kbBacterial artificial chromosomes (BACs) 300 kbPhage P1 artificial chromosomes (PACs) 300 kbYeast artificial chromosomes (YACs) 600 kb____________________________________________________________

•The vector can replicate in either E. coli or mammalian cells.

•It can replicate in E. coli at low copy number under the control of the bacteriophage P1 plasmid replication unit or be amplified by inducing the phage P1 lytic replication unit (under the control of the lac inducible promoter).

•It can replicate in mammalian cells by using the origin of replication (oriP) and nuclear antigen 1 of the Epstein-Barr virus.

Construction and screening of Library

• Why required?• What is this library content?• How to find the book from this library if it is

huge collection of books?

Types of Library

1. Genomic library2. cDNA library

Which library to use?

Genomic Library

Partial digestion

How to do partial digestion?

mRNA

Analysis of RNAs (messenger) by RT-PCR

cDNA library

How to find the gene from the library?

• Selection of the gene of interest

How you will know the plasmid or DNA you chosen the correct one?

• In case of DNA• In case of RNA• In case of Protein

Southern Blot Hybridization

Northern Blot Hybridization• RNA blots are called northern blots in recognition of the fact that the procedure is

analogous to the Southern blotting technique, but with RNA molecules being separated and transferred to a membrane.

Analysis of RNAs by Reverse Transcriptase-PCT (RT-PCR)

Analysis of Proteins by Western Blot Techniques