Strange amplification pattern in DNA real time PCR - (Sep/24/2008 )
I have finished optimizing my SYBRgreen DNA real time PCR and am now working on a probe version (same primers, and probe binds inside amplicon). Perhaps someone might have an idea as to why I am getting linear rather than sigmoidal curves? The anneal temp is as high as it can go for the reaction to work, probe concentration is 100µM, primer concentrations are 375µM, rest is a master mix from supplier. Amplicon length is 130bp and melt time is 20sec, anneal is 30sec. Any ideas?
blue = unknown, red is standard, black is pos control, light blue is no template, and green is neg control. thermocycler is iCycler
Try dilute your DNA template first. Good luck
All DNA template has been diluted to 100ng/ well in 5µl (diluted into Mol grade H20). That should be diluted enough no?
I actually spek'd the template and all but one of those linear samples were between 8-15ng/µl (I added 5µl template to rxn). Only 1 was 28ng/µl which is still low... I ran a gel thinking contamination, but there was none (all neg were neg, and same with positives).
FYI, spoke to tech support, and this is likely a result of not letting hte iCycler warm up long enough, in conjunction with bubbles in the wells. I tried again, and while there were still some bubbles, the next PCR looked better after warming up.