Hi everyone!
My colleague and I, each did a gradient PCR to find the best annealing temperature. Although we used the same set of primers, we got differents temperatures (I got 72 C and he got 64 C)!
Do you have any idea what may cause this difference? I appreciate your help!
What gradient did you run? And what gradient did he run? Are you both interpreting the direction of the gradient on the instrument the same way? Maybe one of you just has it reversed? Usually, in a gradient PCR, there is a trend of product yield and quality as the annealing temperature changes. If the trends that you saw were in opposite temperature directions, there is some serious reagent labeling problem (or the aforementioned error in interpreting the direction of the gradient on the instrument). But sometimes a primer pair will work well all across the range of temperatures tested, so it's possible that this happened in your case and there really is not much difference between 64 and 72.
Different buffer composition could make a big difference... were the reactions done with the same reaction mix?
we are using the same protocol and the same kit, different boxes though (which I don't think it is an issue)
Yeah! Actually, Govinda's message abt the buffer composition made me thinking of taking the buffer from another box as well! I'll let you know the result. Tnx Eman!
What gradient did you run? And what gradient did he run? Are you both interpreting the direction of the gradient on the instrument the same way? Maybe one of you just has it reversed? Usually, in a gradient PCR, there is a trend of product yield and quality as the annealing temperature changes. If the trends that you saw were in opposite temperature directions, there is some serious reagent labeling problem (or the aforementioned error in interpreting the direction of the gradient on the instrument). But sometimes a primer pair will work well all across the range of temperatures tested, so it's possible that this happened in your case and there really is not much difference between 64 and 72.
you do not say what the gradient range is but it does look likely ( as david suggests) that one of you has reversed the samples on the gel...is 72 the same number of samples from the end of the gradient as 64 is from the other end of the gradient?
PCR efficiency is always driven by one of the two primers, the primer with the highest binding efficiency.
In your case it could be that both primers are capable of driving the PCR, but at different temperatures, there might be circumstancial differences in PCR setup.
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