Hi everyone,
I need your help with optimazing my PCR reaction. My PCR product should be 850bp. Starters Tm is 65, they do not form dimers or secondary structures and they’re specific (Blast doesn’t show any unspecific product that may occur). I've tried gradient PCR with typical mix and with Hot start polymerase and every time I receive strong band that is 150bp. What can I do to get rid of this unspecific product and what can it be?
try running the pcr with 6%dmso or final concentration of 1Molar betaine. If any of your colleagues have a polymerase with a high GC buffer then you can use this instead of dmso/betaine. It may be that your template has a high GC or highAT region and is forming a loop that the taq reads across so giving a short amplimer . Sequencing the pcr product would almost certainly show where the problem starts. If your gene has pseudogenes which lack introns then you may be amplifying a short pseudogene product
Sounds like primer dimers (even if you do not think they are that) are the major product you are getting. As Paul Rutland mentioned, I would try DMSO or Betaine. If you do not have quick access to that, try a two part PCR...split your PCR reaction volume (say 50 ul) into two (25 each) where each reaction contains either primer but not both. Let that go for 5 rounds with a 55C annealing temp. After the 5th round mix both reaction into one tube and let it proceed with you 65C annealing temp for another 30-35 rounds. I have had a lot of success with this approach for hard to amplify sequences.
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