Why does the annealing temperature have to be above 55 degrees C, when using the GeneAmp PCR Carryover Prevention kit (Cat. No. N808-0068) or any kit containing AmpErase UNG (Cat. No. N808-0096)?
Uracil-N-glycosylase (UNG) has activity in the temperature range of 4 degrees C to 55 degrees C, and is not completely inactivated by incubation at 95 degrees C. It is recommended that the annealing temperature be at or above 55 degrees C when using this enzyme with any of our PCR or Sequence Detection Systems kits to avoid possible degradation of newly synthesized dU-containing PCR products by residual UNG activity. For this reason, it is also recommended that the post-PCR soak be maintained at 72 degrees C (instead of 4 degrees C) if analysis of the PCR product will be performed using a gel-based method.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.