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Team:Newcastle/PCR
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Revision as of 09:58, 5 August 2010 by Shethharsh08 (Talk | contribs)
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GoTaq PCR
Materials required
Add the following as mentioned below to make up to a final volume of 50 µl in the PCR tube:
- 32.5 µl of distilled H2O
- 10 µl of 5x GoTaq Buffer
- 1 µl of dNTPs
- 2.5 µl forward primer
- 2.5 µl backward primer
- 1 µl template DNA
- 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:
- Initialise - 95°C for 2 minutes.
- Denature - 95°C for 30 seconds.
- Anneal - 52°C for 30 seconds (depends upon the melting temperature, Tm, of template)
- Extension - 75°C for 30 seconds
- Extension finish - 75°C for 5 minutes
- 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 in the PCR tube:
- 27.5 µl of distilled H2O
- 10 µl of 5x Buffer
- 1 µl of dNTPs
- 5 µl forward primer
- 5 µl backward primer
- 1 µl template DNA
- 0.5 µl of Phusion polymerase
Conditions for ThermoCycler
After putting the PCR tubes in the thermocycler, set the thermocycler's condition as mentioned below:
- Initialise - 98°C for 30 seconds.
- Denature - 98°C for 10 seconds.
- Anneal - Depends on the melting temperature, Tm of the primer
- Extension - 72°C (The extension time depends on the the size of the fragment which is to be amplified).
- Extension finish - 72°C for 5-10 minutes
- Hold - 4°C
- An important point to note is that the rate of extension by Phusion polymerase is 1Kb/ 30 seconds and thus always set extension time based on the size of the fragment you are planning to amplify.
Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.
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