missing nt at the 5' end - site mutagensis using NEB kit (Feb/03/2011 )
I really hope you can help me with this issue. I am trying to add 18nt insert into the pmal_p2 plasmid, I designed two primers each with 9 nt overhangs, so basically after ligation I expect 18nt insert. My sequencing results are a bit odd, I am missing nt at the ligation site. I have only 9 nt addition in the insert from one and I am missing the 12 nt that were supposed to come from the other primer.
It seems like one of the primers was incomplete, but this should not happen. Any ideas what could have happened? Is there way to make sure that I am dealing with the full length primer?
I also read that pfu has nuclease activity, could this be a factor?
Your help is highly apperciated,
Oligos are synthesized from 3' to 5', and short oligos are common in typical non-purified preparations. You could HPLC purify them to eliminate short oligos, or simply sample another colony in your transformation, which will likely be correct, or at least different.