To amplify the given sample of DNA using PCR.
Polymerase chain reaction (PCR) is a very simple method for in vitro DNA
amplification using Taq polymerase.
This technique innolves DNA synthesis in 3 simple steps.
Step 1. Denaturation of the template into single strands.
Step 2. Annealing of primers to the template.
Step 3. Extension of new DNA.
Millique water or autoclaved double-distilled water, sterile microfuge (0.2 mL),
10 X Taq polymerase assay buffer with MgCl2, or NTP mix solution, template
DNA, forward and reverse primers, Taq DNA polymerase enzyme, sterile mineral
oil, and a PCR machine.
- Add 38 mL of sterile millique water (or autoclaved double distilled water)
to a sterile microfuge.
- Add 5 mL of 10 X Taq polymerase assay buffer with MgCl2 to the microfuge.
- Add 3 mL of 2.5 mm dNTP mixed solution to the microfuge.
- Add 1 mL of control template DNA.
- Add 1 mol each of forward and reverse primers.
- Add 1–2 units (0.5–0.7 mL) of Taq DNA polymerase.
- Gently mix.
- Layer the reaction mixture with 50 mL of mineral oil to avoid evaporation.
- Carry out the amplification using the following reaction conditions.
- Initial denaturation at 940°C for 1 min.
- Denaturation at 940°C for 30 sec.
- Annealing at 480°C for 30 sec.
- Extension at 720°C for 1 min.
- Final extension at 720°C for 2 min.
The DNA was amplified.