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PCR Profile for ligation - (Oct/28/2013 )

Does someone have a PCR profile for ligation reaction???




You mean a thermal cycler profile?  PCR is polymerase chain reaction, and quite different to ligation.


The profile on a thermal cycler would be dependent on how you want to do your reaction (room temp?, 16 deg? 4 deg?) and how long you want to do your reaction (generally shorter for higher temps).  I generally use 16 deg with a hold until I tell it to stop (usually overnight).


I usually ligate on the bench for 15-30 minutes. Ligations with overhangs almost always work well, without optimization, if the DNA fragments are correct and at the appropriate concentration.


Few years ago I used PCR for ligation step. Now I lost the profile and never did cloning for a long time so forget every thing about cloning now.