GoTaq PCR

Materials required

Add the following as mentioned below to make up to a final volume of 50µl:

1. 32.5 µl of distilled H₂O
2. 10 µl of 5x GoTaq Buffer
3. 1 µl of dNTPs
4. 2.5 µl forward primer
5. 2.5 µl backward primer
6. 1 µl template DNA
7. 0.5 µl of GoTaq polymerase

Conditions for ThermoCycler

After putting the PCR tubes in the thermocycler, set the thermocycler's condition as mentioned below:

1. Initialise - 95°C for 2 minutes.
2. Denature - 95°C for 30 seconds.
3. Anneal - 52°C for 30 seconds (melting temperature, Tm, of template)
4. Extension - 75°C for 30 seconds
5. Extension finish - 75°C for 5 minutes
6. Hold - 4°C

Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.

Phusion PCR

Materials required

Add the following as mentioned below to make up to a final volume of 50µl:

1. 27.5 µl of distilled H₂O
2. 10 µl of 5x Buffer
3. 1 µl of dNTPs
4. 5 µl forward primer
5. 5 µl backward primer
6. 1 µl template DNA
7. 0.5 µl of Fusion

Conditions for ThermoCycler

1. Initialise - 98°C for 30 seconds.
2. Denature - 98°C for 10 seconds.
3. Anneal - x°C for 20 seconds (melting temperature, Tm, of template)
4. Extension - 72°C for 30 seconds per kb
5. Extension finish - 72°C for 5-10 minutes
6. Hold - 4°C

Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.