Adding A overhangs - primer design implications? - (Dec/29/2009 )
I've been having some trouble with adding A-overhangs to my blunt PCR product, so I looked into it. I found this paper (Biotechniques in Vol 20 No. 6, pp1004-10) that says that Taq prefers to add a terminal base adjacent to a C ie a 5'G on your primers and that having a 5' T on the primers pretty much stops the terminal base addition. My primers have a TAAT cap at the 5' (forward primer) and 3' (reverse primer) ends so I couldn't help wondering whether that could be the problem. My gut tells me that I should get some product with As added on both ends, even if the efficiency is not that great.. But no clones so far after ligation in A/T vector.
Thoughts? Have you encountered similar problems or do you think primer design is of relevance when it comes to A-tailing?
very strange - I was under the strong impression that it should work anyway - no matter what nucleotide you have at the end.