Hello, I am trying to amplify cellular (RNase P) and viral DNA at once in a PCR reaction. Both have been optimized to my PCR settings but the RNase P band (the smaller band) has greater intensity than the larger band (viral DNA). The larger band (viral DNA) is very strong in the absence of cellular DNA at the same PCR conditions. Does anyone have any suggestions on how I can increase the amplification of my viral DNA in the presence of RNase P amplification besides doing nested PCR?
In a PCR reaction with two competing amplicons usually the shorter gets a competetive edge. I suggest you optimize your duplex PCR by decreasing the amount of primers for the shorter amplicon and possibly also increase the concentration of the longer. Another thing that could help is to prolong the extension step. This may help if your amplicons are long and/or the size difference is significant. Choice of polymerase could also affect the outcome: Hot Start versions of highly processive polymerases (e.g. Phusion, Q5) would improve the result in most cases.
yep just do pcr which is optimal for amplcion for viral, and u can also try touch down PCR
I have tried increasing the primers for the longer amplicon but it did not increase the amplification of the larger amplicon and have tried gradient PCR. That is a good suggestion I will attempt to decrease the primers for the shorter amplicon and adjust the extension step. Thank you for your help!
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