Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

PCR amplification with new restriction sites troubleshooting - (Apr/15/2013 )

Hi all

I'm using Phusion polymerase to amplify a 4.2 kb target in a pcDNA3 vector. I once used the same template amplify the target successfully in GC buffer and 5% DMSO , annealing temperature 57.5. I'm using new primers to amplify the same template from the same vector and with nearly the same Tms and recognition sequences. But this time I can't amplify the target even using GC buffer and DMSO. The reaction products are high molecular weight nonspecific products that resemble the products i previously got in HF buffer without DMSO.

The Tms for these are 62.9 (50 % GC) and 63.6 (50 % GC). Previous primers that worked were 62.9 (55.5% GC) and 63.7 (55.5 %GC).

Is it worthwhile to use my previous PCR product as template for this new reaction in case there are sites within the vector that are getting amplified giving spurious products? I'm really quite desperate. Help!! :(


I take it you have done Mg2+ and temperature gradients to optimise these primers?


temp grads yes but magnesium no . I didn't do a magnesium grad because teh product is already non-specific and didn't see how magnesium would help increase specificity