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PCR Failure - GA Overhang Addition


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#1 JamASharpe

JamASharpe

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Posted 18 June 2014 - 01:51 PM

Hey all,

 

I'm trying to PCR some overhanging regions into my Vector template for Gibson Assembly.  The purpose of which is to swap out a region of interest for GFP for membrane localisation studies.

 

The 3' end of the vector contains a (G4S)3 linker region which has a very high GC content and the oligo I have to use has a hybridisation region of 80% GC and has a larger than comfortable repeat region of C's.  I am using NEBs Q5 HF master mix and the NEB Tm calculator to try and ascertain the correct Ta for the reaction.

 

All attempts have failed to date.  I have bought 3 different primers for this particular PCR and all have failed, at some expense!  I am now going down the route of doing a gradient PCR from the range 58deg to 72deg.  This will hopefully give a result.  I have also run (in parallel) the same reactions but with the addition of 5% DMSO.  I'm not sure what the DMSO will do in this case though as I'm lead to believe that Q5 HF mastermix already contains 8% DMSO - but I want to try everything before giving up.

 

Does anyone have any experience of trying to PCR GC rich regions with Q5?

 

Many thanks.






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