I'm having a seriously strange issue I hoped someone out there may shed some light on.
I have been for some time attempting mutagenesis and cloning of a rattus norvegicus testes gene into pENTR/D-TOPO. I have been using the Ho method of Mutagenesis (PCR fusion of two fragments of gene created using the forward primer plus a reverse primer that hybridises to the area to be mutated with the site of mutation in the centre of the primer; and vice versa for the reverse plus a forward mutagenic primer); and I have been having great success in cloning the mutagenised products - all sequence confirmed - absolutely perfect. BUT, when I attempt to clone the wild-type (minus any mutation) I have no success whatsoever.
My PCR product is fine - perfect. the CACC region intact
I have even tried using the same internal primer method as the mutagenesis excluding the mutation - no success!
the mutations are single point - ie the only difference between the WT and the mutagenised product is 1-3bp.
I cannot for the life of me figure out what is wrong.
i thought maybe the gene was toxic but thought it unlikely considering that the product with the mutation present (as mentioned only 1-3bp difference to WT) clones perfectly with no drams.
if anyone could offer any insite i would be very grateful.