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need help to evaluate primer sets for SYBR green qPCR - (Aug/30/2007 )


Have designed primer sets for future SYBR green qPCR experiment, just run a conventional PCR with end point electrophoresis. positive control showed one product of expected size, no dimers closedeyes.gif , however, the NTC showed primer dimers around 50bp. blink.gif Since I am still waiting for my qPCR instrument to come, so no further test can be done yet to evaluate primer set. Could anybody tell me whether the dimers mean I am doomed?



Naw, I wouldn't worry about it... if you want to run a neat test try running your NTC without amplification you can see if you are just viewing primers on the gel or if they are really primer dimers (amplification dependent)... No worries if it is only in your NTC, also there is a trick with qPCR where you do the acquisition at 81C this melts primer dimers and prevents them from being counted in the fluorescence, the main thing is if the primer dimers are affecting your reaction efficiency but it sounds like they are not, what I mean to say is - if the primers preferentially interact with one another even when there is template present that may lower the efficiency of your reaction, however in the case where the primers only interact with each other when there is no other choice (template isn't present) then it probably will not affect your reaction efficiency...

HTH and GL!


Thanks for the infor, Beccaf22. Now I can relax a little bit before seeing my supervisor rolleyes.gif