Amplification of CAG promoter problems - (Jan/27/2013 )
I have problem amplifying CAG promoter, apparently its very GC rich some regions have 70% . We've tryied Phusion HS from thermo, Turbo polymerase from Agilent , Taq from NEB, tried different DMSO concentrations, different primers from 55 to 66 C, GC rich buffer, MgCl2 concentrations.
Did anyone have tried to PCR amplify this promoter, would appreciate the help.
How long is your amplicon? Your problem may not necessarily caused by the high GC content. Make sure everything (template, primer, etc) in your system is fine. There are also kits available for amplifying GC rich templates, such the one from Clontech Advantage GC 2 Polymerase Mix & PCR Kit
The product size is less than 2kb, so its shouldnt be a problem, my next step is to try using Platinum plx polymerase from invitrojen that has KOD
Two additional things to try:
Instead of DMSO, try 3-7% of a 1M betaine solution (aka Qiagen "Q-solution").
Try the NEB Q5 polymerase with the GC buffer.
I was curious whether you've figured out a solution? I PCR'd CAG way back when to make a vector, and now I"m trying to PCR it again using the same primers with no luck. I've tried Invitrogens HiFidelity Taq, Phusion, and Q5 with and without GC buffers and with and without DMSO. I'm running low on options.