lec5W11 - Todays lecture Finish restriction enzymes Setting...

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Today’s lecture Finish restriction enzymes Setting up a digest Plasmids ori
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How Sal 1 cuts: 5’NNNN GTCGAC NNNN3 3’NNNN CAGCTG NNNN5’ After Sal 1: 5’NNNN G TCGAC NNNN3’ 3’NNNN CAGCT G NNNN5’ 5’NNNN G -OH P- TCGAC NNNN3’ 3’NNNN CAGCT -P OH- G NNNN5
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How often will RE cut a sequence? A four base recognition sequence should cut once every 4 4 nucleotides, assuming there are equal numbers of A,C,T,and Gs. So average size of pieces would be 256 bp. A six base recognition sequence should cut once every 4 6 nucleotides, with average size of pieces 4096 bp.
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Sal l and Vibrio DNA Sal l has a six base recognition sequence which is dominate by GCs G}TCGAC CAGCT{G Vibrio genomic DNA is AT rich (62% AT versus 38% GC) – so will average size of fragments be larger or smaller than 4096 bp?
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Conditions for restriction enzyme digests DNA has to be clean – no organic reagents that could inhibit enzyme activity All REs come in glycerol to prevent freezing – want glycerol less than
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This note was uploaded on 04/10/2011 for the course BIMM 101 taught by Professor Butler during the Spring '08 term at UCSD.

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lec5W11 - Todays lecture Finish restriction enzymes Setting...

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