I have the problem of appearance of nan-specific bands when I change the volume of DMSO, Pfu, Polymerase buffer and or DW in sample solution for PCR. I also change the annealing temperature due to impact of DMSO volume.
What could be the possible reason? How can I resolve the issue to have no nonspecific bands?
Hi there,
In a given PCR mix recipe, buffer volume and polymerase volume have to remain constant. In your case only volume of DMSO and volume of water should vary. If you want to be able to interpret what is going on in your set of experiments you should change only one parameter at the time. If you increase DMSO concentration and diminish annealing temperature at the same time, you may see no improvment towards the experiment done without DMSO but with higher annealing temperature.
Dear Najmul,
Usually DMSO reduces the nonspecific bands. If you have so many non specific bands in a pcr product,adding DMSO will reduce the appearance of nonspecific bands. But try to standardise it with different concentration of DMSO.
Hi there,
In a given PCR mix recipe, buffer volume and polymerase volume have to remain constant. In your case only volume of DMSO and volume of water should vary. If you want to be able to interpret what is going on in your set of experiments you should change only one parameter at the time. If you increase DMSO concentration and diminish annealing temperature at the same time, you may see no improvment towards the experiment done without DMSO but with higher annealing temperature.
the annealing temperatures of primers are very dependent on salt and primer concentration so changing any parameter that changes the final salt concentration in the pcr reaction may cause changes particularly if the annealing temperature and specificity of the primers drop then there will be more bands. As Dominique says change only one thing at a time. DMSO has the effect of increasing the annealing temperature and,therefore, specificity of primers. I would run a gradient of annealing temperatures in presence of DMSO and then stay as close as possible to the best conditions for all future work with these primers.
Hello,
According to my experience, if you have a couple of primers which tend to produce multiple bands, sometimes it is more efficient to start from scratch and go for new primers. The optimization of an amplification protocol can taka a lot of time, and not always will you obtain complete reproductibility (due to phenomena such as reaction meta-stability).
I do not have direct experience with DMSO. If I were to modify the protocol I would rather try to work on annealing temperatures, possibly by iuntroducing some touchdown steps at the beginning.
Best regards,
Marco
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