University:
York University
Course:
BIOL 107 | Introduction to Cell Biology
Academic year:

2024
Views:

188

Pages:

2
Author:

Ciara T.

Biotechnology To clone DNA Recombinant DNA must be created. "DNA from 2+ different sources that are joined together" Bacterial plasmid a "gene of interest" gene you would like to study within a particular genome Small double stranded molecule in bacteria Antibiotic resistance Replicate independently Plasmid pBR322 08312: Several unique RE Small size (4.1kb) Genes can be inserted into plasmids for cloning Plasmids serve as cloning vector Lysis DNA is harvested by breaking the cell open Restriction enzyme •Cleaves at restriction sites. multiple cloning site (MCS) occur once/plasmid •Can produce sticky or blunt ends Use Restriction Endonucleases (RE) and DNA Ligase to cut and paste DNA Bacterial enzyme that cuts DNA at specific base sequence Joining of DNA fragments is promoted by sticky ends ↑ Restriction site •Short, specific Palindromic; reads the same on both template strands. Blunt = 5'-GATATC-3' CTATAG STICKY ENDS ON PLASMID AND GENE ARE COMPLEMENTARY ↑ DNA Ligase connects the 2 pieces of DNA permanently G AATTC G G AATTC G CTTAA Sticky ends/ Sticky ends cut, target gene obtained, joined with DNA ligase. Through the process of ligation, recombinant plasmids can be formed. Introduced into host cell (E. coli) for propagation (cloning). The DNA is harvested by breaking the cell open through lysis How to obtain target gene PCR •Denaturation - separate strands in double helix Annealing - attaches primers to DNA