I am using Brilliant III qPCR mastermix to amplify a 60 bp oligo and getting spurious bands on 12% PAGE when I try to amplify using 95 degree denaturation for 15 s followed by 60s extension for 33s (minimum time possible in ABI 7800 system for data collection). I have also used a 62 and 72 degree celcius for annealing and extension for 10 and 40 s respectively. However when I run it in 12 % Gel, I get a band near the wells and a smear. Previously I got amplification when I used Takara polymerase (conditions were 95 (10s), 62(10s) and 72(1 min) degrees for denaturation, annealing and extension respectively in endtime PCR but am unable to replicate it in qPCR. P.S. An amplification is apparent in the qPCR. Can anyone suggest how to troubleshoot this?
Perhaps doing a denaturation step with SDS added into your loading dye will help? I used to do this all the time to remove any proteins that are stuck onto my DNA
Thank you Steve for the input! A quick update: The problem seems to be solved by lowering the amount of primer used as well as the annealing temperature. A lower annealing temperature equal to the melting temperature of the primer seems to be working good. Also the bright band near the well has disappeared with the specific amplification. However, the bands can be seen in no-template controls.
Are you sure those bands in the no-template controls are not contaminations? Best
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