34_Lieb_Sequencing_PCR

34_Lieb_Sequencing_PCR - Recombinant DNA IV: PCR and DNA...

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Unformatted text preview: Recombinant DNA IV: PCR and DNA Sequencing J. Lieb Review of Last Lecture Screening Colonies- Colony lifts, transferred to membrane and probed Southern blot – DNA on gel, transferred to membrane, and probed. Northern blot – RNA on gel , transferred to membrane, and probed. Western blot – proteins on gel, transferred to membrane, and probed. RFLPs (R estriction F ragment L ength P olymorphisms) can be useful markers for genetic variants or disease PCR The P olymerase C hain R eaction allows in vitro amplification of any DNA sequence PCR amplifies DNA without in vivo cloning 5’-GGGCCATATGGTCCAGTCACCCAAGGTCAGTCCAACGGACTTAGTATTGCACCGAC-3’ 3’-CCCGGTATACCAGGTCAGTGGGTTCCAGTCAGGTTGCCTGAATCATAACGTGGCTG-5’ template 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GTCGGTGCAATACTAAGTC-3’ primer 1 primer 2 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GGGCCATATGGTCCAGTCA-3’ How does it work? 5’-GGGCCATATGGTCCAGTCACCCAAGGTCAGTCCAACGGACTTAGTATTGCACCGAC-3’ 3’-CCCGGTATACCAGGTCAGTGGGTTCCAGTCAGGTTGCCTGAATCATAACGTGGCTG-5’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GGGCCATATGGTCCAGTCA-3’ DENATURE cycle 1 Denaturing is accomplished by heating the DNA to 95 degrees C 5’-GGGCCATATGGTCCAGTCACCCAAGGTCAGTCCAACGGACTTAGTATTGCACCGAC-3’ 3’-CCCGGTATACCAGGTCAGTGGGTTCCAGTCAGGTTGCCTGAATCATAACGTGGCTG-5’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5 ’- G T C G G T G C A A T A C T A A G T C- 3 ’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GTCGGTGCAATACTAAGTC-3’ 5’-GGGCCATATGGTCCAGTCA-3’ ANNEAL cycle 1 Primers are annealed by cooling the solution to 55 degrees C 5’-GGGCCATATGGTCCAGTCACCCAAGGTCAGTCCAACGGACTTAGTATTGCACCGAC-3’ 3’-CCCGGTATACCAGGTCAGTGGGTTCCAGTCAGGTTGCCTGAATCATAACGTGGCTG-5’ 5’-GGGCCATATGGTCCAGTCA CCCAAGGTCAGTCCAACGGACTTAGTATTGCACCGAC-3’ 5’-GGGCCATATGGTCCAGTCA-3’ 5’-GGGCCATATGGTCCAGTCA-3’...
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This note was uploaded on 06/27/2011 for the course BIOL 202 taught by Professor Kieber-hogan during the Spring '08 term at UNC.

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34_Lieb_Sequencing_PCR - Recombinant DNA IV: PCR and DNA...

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