So I'm trying to PCR homologous arms for CRISPR/Cas9-mediated knock-in. I'm usinga guide RNA that has been previously validated in the Bl/6 background. Unfortunately, they didn't do knock ins, just gene dispruption, so there was no PCRing of homologous arms in the earlier paper.
Here's my dilemma. Using sequence data from the black 6 genome from ENSEMBL, I was easily able to PCR the 3' arm of rosa 26 (~1kb) - line 1. I also wanted to confirm that the guide RNA site was in the genome of the mouse line I'm working with since it's not Bl/6. The primer set for this was a forward primer ~150bp upstream of the putative guide RNA site (line 2). This worked 100% and the sequence was 100% identical to the black 6 genome. Now I just need to PCR the 5' arm. I've tried at least 10 different forward primers to make a 1kb arm (line 3), a 750 bp arm (line 5) and most recently a 500bp arm (line 7). The variations I tried are shown in the attached image; green indicating successful PCRs and red are unsuccessful. I am running out of options and I don't want to make my arm too short (not really sure how short I can go). I tried using Phusion polymerase (GC and regular buffer, with and without DMSO) and Invitrogen's high fidelity polymerase. I generally just get multiple bands but those that are in the predicted length I tried sequencing and nothing is correct. Any suggestions?
Edited by Ahrenhase, 10 May 2014 - 01:39 PM.