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?