PCR STEPS Flashcards
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Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence
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Next, denature the DNA to single strands at 94˚C
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Rapidly cool the DNA (37-65˚C) and anneal primers to complementary single strand sequences flanking the target DNA
4/7
Extend primers at 70-75˚C using a heat-resistant DNA polymerase such as Taq polymerase derived from Thermus aquaticus
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Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (1x10^6) to 35 trillion (35x10^13) copies of the target DNA
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Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely
7/7
Cool to 4˚C and store (-80°C ideally) or use amplified PCR product for analysis
