DNA Amplification by the PCR Method

To amplify the given sample of DNA using PCR.

Principle
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.

Materials
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.

Procedure

1. Add 38 mL of sterile millique water (or autoclaved double distilled water) to a sterile microfuge.
2. Add 5 mL of 10 X Taq polymerase assay buffer with MgCl₂ to the microfuge.
3. Add 3 mL of 2.5 mm dNTP mixed solution to the microfuge.
4. Add 1 mL of control template DNA.
5. Add 1 mol each of forward and reverse primers.
6. Add 1–2 units (0.5–0.7 mL) of Taq DNA polymerase.
7. Gently mix.
8. Layer the reaction mixture with 50 mL of mineral oil to avoid evaporation.
9. Carry out the amplification using the following reaction conditions.
10. Initial denaturation at 940°C for 1 min.
11. Denaturation at 940°C for 30 sec.
12. Annealing at 480°C for 30 sec.
13. Extension at 720°C for 1 min.
14. Final extension at 720°C for 2 min.

Result
The DNA was amplified.