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
• 18.5ul ddH2O

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
• 92.5ul ddH2O

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.