We used DNA polymerases Accustart and Phusion to amplify a gene region about 1Kb long. For this particular DNA template, it has not been successful, despite many tweaks we made to the PCR conditions.
From our experiments, DNA dilutions of 1/25 and 1/50 seem to be better, with the combination of BSA and Mg2+. A very faint band has been produced but not good enough yield.
We have tried different annealing temperatures, 50°C, 46°C, 48°C.
Would like to know if lowering the extension temperature would help to increase PCR product yield, and how.
Thank you.
many times, increase the annealing temperature would help the yield if the priming are correct. change extension temperature has little effect regarding to 1 kb amplicon.
I would recommend a touchdown PCR to reduce nonspecific binding or a gradient PCR to find your ideal temperature. You may also want to check you DNA samples are clean or if any primer interactions.
It seems the problem is in your primer design. Did you check for secondary structure of your primer(s)? What is the calculated Tm?
lowering the extension temperature would not help you to increase PCR product yield, touchdown PCR technique or increasing PCR cycles (2-3 cycles) may help .....good luck
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