Lab 5[1] - Restriction digest ligation and agarose gel...

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Restriction digest, ligation, and agarose gel electrophoresis of bacteriophage λ DNA 1
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Restriction digest, ligation, and agarose gel electrophoresis of bacteriophage λ DNA University of Texas at Austin Department of Chemistry and Biochemistry Introduction The use of DNA ligase allows the reassembly of DNA genomes that have been disassembled by restriction enzymes. The objective of this lab is to digest a bacteriophage λ with two different restriction enzymes, EcoRI and HindIII. Then we will reassemble the λ genome using the enzyme DNA ligase. Upon reassembly, we will analyze the genome using agarose gel electrophoresis by comparing it to a 1kb DNA ladder 1 . Restriction enzymes are naturally occurring enzymes that cut DNA at a certain sequence. They are used to excise a gene from genomic DNA for cloning, opening a cloning vector for gene insertion, adding linkers to DNA for vector insertion, or for genetic analysis using restriction sites as markers. When restriction enzymes cut DNA at a certain sequence, they cut two ways: one is every 4-base or 6-base nucleotide sequences which creates sticky ends and the other method is by creating blunt ends 2 . Once there are two blunt or sticky ends, DNA ligase is used to join the two
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This note was uploaded on 02/14/2010 for the course CH 369T taught by Professor Mcdonald during the Spring '09 term at University of Texas.

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Lab 5[1] - Restriction digest ligation and agarose gel...

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