2. First determine the reverse complent sequence to this strand of DNA. Then design primers (shown in red) that will bind to the 3´ end of each strand of DNA.
3´ GGCATCGACCTCCCT 5´ 5' GGATCGATCAAGAACAATGACAGGATCGAGGAATTCAGCCTACGCAGCCCGTAGCTGGAGGGA 3'
3´ CCTAGCTAGTTCTTGTTACTGTCCTAGCTCCTTAAGTCGGATGCGTCGGGCATCGACCTCCCT 5´ 5´ GGATCGATCAAGAAC 3´
What other reagents are necessary to perform a PCR reaction?
Taq polymerase, nucleotides (dNTPs) and a buffer
PCR machines cycle between three temperatures. What is the purpose of each stage in the PCR cycle, and roughly what temperatures are used?
94oC - denatures template DNA
50-55oC - primer anneals to template DNA
72oC - Taq polymerase elongates DNA
After 10 rounds of amplification how many copies of the amplified region should you have theoretically.
Product is formed in the third round, 2 molecules of product (=21). After that it should double every round for the next seven rounds. Thus you would expect a total of 28=256 molecules. Note that you will continue to make more of the heterogeneous products each round, so this is really an underestimate.