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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-

I have recently extended the homologous targeting arms in my donor template, and now I am encountering trouble with Rosa26 amplification similar to what you described.  

 

My last attempt was using Phusion with GC buffer and 3% DMSO and it failed to amplify from wildtype templates.

 

In your last post, you said you fixed it by using 15% DMSO, but did not mention which buffer was used along side.

 

Would you mind summarizing your final working PCR reaction conditions?

 

Thanks

 

 

 

 

-Theevl-

The 5' side of the ROSA26 contains a good deal of SINE repeats and others. I'd guess this may be at least part of your issue. Have you made sure that your primers don't sit right in the middle of one of these repeats (I'm sure you have)? Other than that, there's not much you can do about them that I know of other than trying various conditions and praying to the PCR gods.

-Bio-Lad-

I have recently extended the homologous targeting arms in my donor template, and now I am encountering trouble with Rosa26 amplification similar to what you described.  

 

My last attempt was using Phusion with GC buffer and 3% DMSO and it failed to amplify from wildtype templates.

 

In your last post, you said you fixed it by using 15% DMSO, but did not mention which buffer was used along side.

 

Would you mind summarizing your final working PCR reaction conditions?

 

Thanks

 

 

 

 

 

I used the regular Phusion buffer, not the GC.  

-Ahrenhase-