Amplification of CAG promoter problemsno product
Posted 26 January 2013 - 11:04 PM
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.
Posted 27 January 2013 - 12:26 AM
Posted 27 January 2013 - 12:34 AM
Posted 27 January 2013 - 07:32 AM
Instead of DMSO, try 3-7% of a 1M betaine solution (aka Qiagen "Q-solution").
Try the NEB Q5 polymerase with the GC buffer.
Posted 13 August 2014 - 08:54 PM
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.