PCR conditions will vary depending on the PCR polymerase and PCR buffer. The control reaction was optimized for Taq DNA polymerase with ThermoPol buffer (NEB#M0267). If using another polymerase or buffer it may be necessary to optimize the reaction conditions. The forward and reverse control PCR primers, the L1 Primer Mix, come pre-mixed at a concentration of 20 µM of each. It is important to run a negative control at the same time as the repair reaction. For the negative control add 1 µL of H2O instead of the 1 µL of PreCR Repair Mix at the appropriate time. This is the unrepaired DNA reaction for comparison.
The following protocol is for a 50 µL PCR reaction.
- At room temperature, combine 38 µl H2O, 5 µl ThermoPol Buffer, 0.5 µl 100X NAD+, 0.5 µl of 10 mM dNTPs, and 3 µl of the supplied UV damaged Lambda DNA.
- Add 1 µl PreCR Repair Mix and mix by gently pippeting up and down 3 times.
- Incubate the repair reaction for 15 minutes at 37°C.
- Place the reaction on ice.
- Directly to the repair reaction, add 1 µl of the control primers, 0.5 µl of 10 mM dNTPs and 0.5 µl of Taq DNA polymerase (5 units/µl, not supplied). Mix gently.
- Place into a thermocycler running the following program once the block temperature reaches > 90°C. Thermocycler program; 2 min at 95°C for 1 cycle, then 10 sec at 95°C, 30 sec at 65°C, 1 min at 72°C for 25 cycles and finally a 4°C hold. A PCR product of 1 kb should be seen.