Premix vendor dependent disssociation curves? - (Feb/03/2010 )

Hi all, recently I received a free sample of a SYBR PCR mastermix from one vendor and I decided to compare its performance with the one I normally use. I set up a reaction and prepared a mastermix of primers and template so that the only different thing between both reactions would be the SYBR premix and I ran them by triplicates. Ct values for the new premix were one cycle smaller than the one I used to use, this really did not surprised me much, but what did surprise me is that in the dissociation plots, each premix produced a peak at different temperatures (single peak, 4-5 degrees C apart from each other). I ran a gel and the products look the same size.

I am sending the products for sequencing tomorrow, since I am really interested in testing the performance of the new premix since it is way cheaper than the one I normally use, but I am worried about the higher temperature peak it produced.

I was just curious to know if anybody of you have any idea of why amplification using exactly the same primers and template but different SYBR premix could generate dissociation plots that indicate amplification of products of different sizes?

Thanks,

E.

Annealing temperature depends on salt concentration as well as GC content. The buffers for the two PCR mixes likely have significantly different salt concentrations. You could experiment with this by adding salt to a pcr product and observe the shift.

phage434 on Feb 4 2010, 07:04 AM said:

Very insightful, I never thought about that, I will try it. Thank you!