PCR

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PCR(Polymerase Chain Reaction) is a basic biological technique which amplify double strain DNA by magnitudes.

Preparation

Thermal cycler(also called PCR machine, DNA amplifier), PCR tubes(200μl capacity), DNA polymerase, dNTP, DNA templates, DNA primers.

PCR Protocol

1.Mix the DNA polymerase, dNTP, DNA templates and DNA primers in a clean PCR tube.

Then put the tube in PCR machine and begin setting:

2.Pre-denaturation: 95°C for 10min;

3.Denaturation:  95°C for 30s;

4.Anealing: 55°C±10°C (The exact temperature is determined by the length and GC percentage of your primers. You could find an answer in the user manual attached with your purchased primers);

5.Elongation: 72°C (The exact temperature is determined by the DNA polymerase you will use. You could find an answer in the user manual attached with the DNA polymerase you have purchased);

6.Cycle: Set the PCR machine to cycle from step 2 to step 4 between 20 and 30 times;

7.Final elongation: 72°C for 10-15min (The exact time is determined by the length of the double strain DNA you want to amplify. Typically, a longer DNA you want to amplify, a longer time you will need to set in this step);

8.Hold: 4°C  for a long enough time untill you proceed using PCR products in the next experiment.