RACE product cloning in TOPO vector - (Nov/20/2006 )
I am trying to clone 3' and 5' RACE fragments in the TOPO vector.
I amplified fragments by RACE PCR with a proofreading polymerase, I purified and A-tailed the fragments and then I cloned them into the PCR4 TOPO vector from invitrogen. I checked the clones by PCR using the T3 T7 primers. The PCR shows the presence of an insert in all the clones. The problem is that when I use my specific primers on the clones I have no amplification, although I have good amplification with the same specific primers on the PCR product before cloning.
Does anyone have an idea of what is going on in this cloning experiment. What can I do?
Thanks for your answers
I don't know what is going on, but the easiest way to figure out is to sequence a few of your clones with T3 and T7 primers and see what is there. Either before or in parallel, you might want to digest some of your DNA with flanking cutting enzymes to make sure your plasmid is uniform and has an insert of the expected length.