me in increments of 1 minute. For PCR using genomic DNA, follow suggestion number 15, below. 12. Insufficient starting template Perform a second round of PCR using a nested PCR approach (see appendix). 13. Primer design not optimal Review primer design (see appendix). 14. RT reaction error For RT-PCR, take into consideration the efficiency of the reverse transcriptase reaction, which averages 1030%. The added volume of reverse transcriptase reaction should not exceed 10% of the final PCR volume (see appendix). 15. PCR of long fragments
from genomic DNA When amplifying products longer than 4 kb from genomic DNA, increase the concentration of genomic DNA in the reaction (see appendix). Alternatively, use the protocol for amplification of long PCR products using ProofStart DNA Polymerase and QIAGEN Taq DNA Polymerase (see the Taq PCR Handbook, March 2002, or the ProofStart PCR Handbook, February 2002). 16. PCR overlaid with mineral oil when using a thermal cycler with a heated lid When performing PCR in a thermal cycler with a heated lid, do not overlay the PCR samples with mineral oil if the heated lid is switched on as this may decrease
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