In this procedure the PreCR repair and PCR reactions are performed in the same tube. This is convenient if using ThermoPol buffer for the subsequent PCR reaction. Other PCR buffers may work in the repair reaction. If using a buffer other than ThermoPol and optimal repair is desired, then the Sequential Reaction Protocol is recommended (see below).
The following protocol is recommended for a 50 µl reaction volume:
- At room temperature, combine 1X ThermoPol Buffer, 100 µM dNTPs, 1X NAD+, damaged template DNA and H2O to 46 µl.
- Add 1 µl of the PreCR Repair Mix, and mix gently.
- Incubate the repair reaction for 15-20 minutes at 37°C.
- Place the reactions on ice.
- Add the primers, a second aliquot of dNTPs (another 100 µM) and the PCR polymerase directly to the repair reaction mix.
- Proceed with the PCR amplification protocol.
Notes: The extent of damage that a particular DNA template contains is variable. Therefore, it is advisable to try different amounts of template in the amplification reaction. If possible, a titration range of 200, 100, 50 and 25 ng of template DNA in the amplification reaction is recommended.
For some types or extents of damage, optimal repair occurs at 4°C and a 15 minute incubation. In certain cases we have found that a 4°C overnight incubation is best.
Incubating the repair reaction at 37°C is recommended, however, a room temperature incubation will work nearly as well.
As with all PCR reactions, conditions may need to be optimized to achieve maximum amplicon yield.