Lab7_Wilkerson - Christopher Wilkerson 1 6/24/08...

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1 17:36:52 Restriction Enzyme Digest & Agarose Gel Electrophoresis Objective : The purpose of this lab is to digest large samples of both promoter and gene plasmid DNA with restriction enzymes that will allow the gene to later be placed downstream of the promoter in a new plasmid. These cleaved fragments will then be separated out using agarose gel electrophoresis. Restriction enzymes are proteins that recognize specific sequences in DNA and cleave the chemical bonds that hold the DNA together. Two types of cleavages can be produce from this interaction; sticky and blunt. “Sticky” cleavages leave overhangs on the ends of the DNA. These palindromic overhangs are sequences of DNA no longer base paired to another strand of DNA that read the same to their complementary sticky end. The meaning of palindromic in relation to genetics is exemplified by this sequence: GTAC has a complementary strand reading CATG . However when reading both in the 5’ to 3’ direction assuming that the first sequence was in this order, they are one and the same: GTAC . This variety of cleavages is favorable because when ligase is used to join two stands of sticky DNA only compatible strands, made so by their specific base pairing, will be able to be spliced back together. This allows calculated assembly of new plasmids that will contain the gene combinations in precisely the desired order. Blunt ends have no overhang and therefore are compatible to be ligased together with any other blunt ended DNA. However, the non-specific nature of blunt ends is not desired for this experiment. Figure 1. Examples of cleavage sites.
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Lab7_Wilkerson - Christopher Wilkerson 1 6/24/08...

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