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Team:Imperial College London/Wetlab/Protocols/PCR
From 2009.igem.org
Contents |
PCR Protocol
Reaction Mixture
Each PCR tube should contain: (25ul total)
- 2.5ul of 10x Pfu Ultra Buffer
- 0.5ul dNTPs
- 1ul Primer Fwd (100ng)
- 1ul Primer Rev (100ng)
- 0.5ul Pfu Ultra
- 1ul DNA Template
This can be made as a master mix, to reduce variation between reaction mixtures. Master Mix contents (for 4 PCR tubes):
- 12.5ul Pfu Buffer
- 2.5ul dNTPs
- 5ul Primer Fwd (500ng)
- 5ul Primer Rev (500ng)
- 2.5ul Pfu Ultra
Pipette 24ul of the master mix into different PCR tubes. To each experimental tube add 1ul DNA template, and to the negative control, add 1ul ddH2O.
Cycles
X = Annealing Temperature - depending on primers (X1 temp without overhang, X2 temp with overhang), Y = Length of contruct to work out elongation time (for Pfu Ultra II, the rate is 15sec per kB, minimum 15 seconds)
1 cycle: 2 minutes at 95'C
10 cycle: 30 seconds at 95'C, 45 seconds at X'C, Y seconds at 72'C
20 cycle: 30 seconds at 95'C, 45 seconds at X2'C, Y seconds at 72'C
1 cycle: 5 minutes at 72'C
Tips
- Use master mixes. They reduce the variation between reaction mixtures.
- Negative control has no DNA template in it.
- Vary the first annealing temperature across a gradient (usually in 2'C intervals), and keep the second annealing temperature constant. The temperature of the negative should always be at the lowest temperature of the reaction.
