BIOB12H3 Lecture Notes - Lecture 5: Restriction Enzyme, Subcloning, Restriction Digest
Document Summary
Molecular biology module: restriction enzyme analysis and agarose gel electrophoresis. Laboratory 5a: restriction enzyme digest of plasmid dna isolated in lab. In today"s lab, you will set up a restriction enzyme digest on an aliquot of your plasmid dna that you purified in lab 4b and separate the product of digestion by agarose gel electrophoresis. Restriction enzymes are found in nature in prokaryotes. Molecular biologists have cloned the genes encoding these restriction endonucleases and hundreds of these enzymes are now readily available from commercial sources. Generally, these enzymes require a specific buffer and salt (such as. Mgcl2) concentrations and can take approximately one hour to digest 1 g of dna to completion. The type ii restriction endonucleases recognize short, palindromic sequences and make two single stranded cuts at the target site. These may generate 5" overhangs, 3" overhangs or blunt ends. Examples of each of these are shown below:
