TA cloning: if PCR product is not freshly prepared - (Mar/10/2006 )
As we know that for TA cloning PCR product should have A overhangs when we amplify it with taq poymerase and the amplified product should not be very old as with time these overhangs might be lost. But can we find out whether these overhangs are lost or not by just checking the PCR product on the gel? Because if we have RT-PCR product and we do not have anymore cDNA left for further amplification and we do not want to go back to the RNA step then what should we do?
I read a recipe were you would just mix your DNA with Taq and buffer at 72 ºC for 5 to 10 minutes, and this was supposed to add the overhangs. It was from a TA cloning kit from invitrogen I think, but I just can't put my hands on it. Sorry.
i agree with canalon.
My pcr was old (but stored at -20°). I added 1µl Taq and heat 72° 10'. Then standard Topo reaction. If pcr product is purified, add the required amount of taq buffer, nucleotides etc... and proceed same way.