General PCRs: Protocol

1. Retrieve a sample containing a nucleotide sequence (can be from hair, blood, skin, etc. or from a known organism).
2. You will also need DNA primers. These are short single stranded DNA that attach to nucleotide sequences, thus creating a complementary strand of nucleotides.
3. DNA polymerase is another necessary element in a PCR. This enzyme is very sensitive to temperature, so it should only be taken out of the freezer when needed. The enzyme attaches the nucleotides to form a complementary base pair, synthesizing a full complementary nucleotide strand of DNA. Though there are many different types of DNA polymerases, the one most commonly used is Taq polymerase, because it is derived from heat-resistant bacteria, and improves the ability to perform a PCR (which involves many temperature changes). Taq is also a cheaper type of polymerase, so it is a better choice to start with.
4. An excess of nucleotides will also need to be placed into the mixture, so that the polymerase has something to work with. Nucleotides contain Adenine, Thymidine, Cytosine, and Guanine (A, T, C, G).
5. The DNA segment (can be diluted) is placed in a tube containing the items above (be sure to add the Taq polymerase last, so it will be more effective). The tube will then be placed in a thermoregulator, which can easily adjust temperatures. The solution is first heated to at least 94C. This step breaks the hydrogen bonds, thus allowing the strands to separate. This is called denaturation. Then, the tube is cooled to about 54C; at this temperature, the DNA primers start biding to the single stranded DNA. At 72C, the process of DNA polymerisation increases rapidly, creating the double stranded DNA molecules.
6. The cycle can be repeated multiple times depending on the quantity of DNA needed. The most common number of cycles is 30 cycles. Since each cycle doubles the DNA once, 30 cycles results in 2^30 samples.