I expect a 70 bp amplicon which shows up only with 2% (or higher) DMSO in the PCR. But with DMSO, the size of this amplicon goes from 70 bp to ~110b bp on 3% agarose gel. Same happens when a positive control PCR is done using same primers. In the positive control too, the size goes from 70 bp to ~110 bp when done with 2%DMSO. Is this size shift common if DMSO is added in to PCR?
Hi Sanjay, DMSO and other additives not only facilitate strand separation but also lowering primers annealing temperature. Each additive has their role in different PCR products. Some additives works better in specific a PCR reaction which fails for other reactions. In my experiment using 3-5%DMSO, there is no effect on the amplicon size. In your experiment you need firstly to recalculate primer Tm values for correct annealing temperature (put in consideration DMSO lowering primers annealing temperature). Also you should verify that primers have no additional complementary regions within the template DNA and adjust Mg ++ concentration.
regards
Already tried temperature gradients from Tm-10 0C to Tm+10 0C, one PCR tube every 2 0C, the size remains same ~100 bp instead of 70 bp without DMSO. Also tried varying MgCl2 but no improvement. I am going to sequence this 100 bp to see what exactly is it.
It could be a contamination. Sequencing is the best solution. I hope you will share the result as well.
Zhao, the sequencing results turned out to be perfect. The 100 bp fragment on the gel turned out to be 70 bp by sequencing. My guess is the issue was electrophoresis since I was using ~4% agarose gel which might have caused the shift, though I did all the non-DMSO controls on the same gel which were 70 bp.
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