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Troubles with PCR (Rosa26 locus in mouse line) VERY CONFUSING! - (May/10/2014 )

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?


Attached Image

-Ahrenhase-

I solved the issue by using 15% DMSO and annealing time of 5 seconds.

-Ahrenhase-

So it must have some pretty strong secondary structure in there then?

-bob1-

So it must have some pretty strong secondary structure in there then?

 

At first I thought it was nonspecific binding to other regions of genomic DNA but then I fortuitously found a vector containing Rosa26 regions and my amplicon of interest.  Again, I got multiple bands.  So then I just set up at least 10 different reaction conditions until I found a formula that worked. 

-Ahrenhase-