Reactions of digestion

Reactions of digestion - Reactions of Restriction Digestion...

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Reactions of Restriction Digestion and Ligation Paresh Jha 4/14/08 Section 6
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The goal of this lab is to simply understand the process of restriction and ligation reactions through the process of agarose gel electrophoresis. At the forefront of this process are substances known as restriction endonucleases. These enzymes recognize specific palindromic sequences in DNA and cut at those sites. They are found in the immune system of bacteria. Palindromic sequences are nucleotide sequences that are read the same in the 5’ to 3’ direction on both strands. Once these sequences are cut it can also be ligated back together or in different ways. Ligation of DNA fragments by DNA ligase is an essential step in many molecular biology techniques including, gene cloning and messenger RNA fingerprinting. The T4 DNA ligase is the one used in this experiment and it is originally found in Escherichia Coli . It puts the sequences back together by forming a phosphodiester backbone between 2 nucleotide bases. During gel electrophoresis, DNA fragments cut from a sample move through a gelatinous medium composed of agarose. The gel is cast as a thin slab, with a series of small wells at one end. Sample fragments are placed in the empty wells alongside one that gets filled with a marker, sometimes called a DNA ladder. The marker contains DNA fragments whose base sequences and lengths are already known. It is used to help estimate the size of unknown DNA fragments. In the case of this experiment, a lambda DNA fragment is used as the control and reference or the other restriction and ligation reactions in the different lanes. The gel then gets placed in a chamber filled with a liquid buffer that can carry an electrical current. This lab involved three restriction digestion reactions along with one ligation reaction.
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This note was uploaded on 09/16/2008 for the course MCB 213 taught by Professor Zhang during the Spring '08 term at UConn.

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Reactions of digestion - Reactions of Restriction Digestion...

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