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Lecture 5-LC710 1st hr Posted

Lecture 5-LC710 1st hr Posted - 710.LC GRADUATE MOLECULAR...

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710.LC GRADUATE MOLECULAR BIOLOGY 9/15/2010
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Lecture 4 Competency Test.
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Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Palindrome Restriction site Fragment 1 Fragment 2
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5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts
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Common Restriction Enzymes E co RI Eschericha coli – 5 prime overhang P st l Providencia stuartii – 3 prime overhang
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1) Name the five components of a PCR reaction. 1) Template 2) Buffer 3) Primers (two of them) 4) Taq Polymerase 5) dNTPs
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The PCR Reaction How does it work? Heat ( 94 o C ) to denature DNA strands Cool ( 52 o C ) to anneal primers to template Warm ( 72 o C ) to activate Taq polymerase, which extends primers and replicates DNA Repeat 35 cycles
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Denaturing Template DNA Heat causes DNA Heat causes DNA strands to separate strands to separate 3’ 5’ 5’ 3’ Denaturation of Denaturation of DNA at 94 DNA at 94 o C 5’ 3’ 3’ 5’
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