AT rich PCR problems - anyone experienced with tetramethylammonium chlroide? (Feb/28/2007 )
I've been having some issues trying to amplifly a ~2.5kb region thats 70% AT. There is a paper (Chevet et al 1995. Nuc Acid Res. 23(16) 3343-3344.) that shows by using 60mM tetramethylammonium chloride they can increase yield because it causes the AT bonds to become more stable. I've also seen some suggestions of lowering the extention temperature to about 62 degrees.
Does anybody have any experience (good bad or ugly) with either of these situations?
I never used the tetramethylammonium chloride but was very succesfull decreasing the extension temperature. I would suggest to try a gradient PCR with extension temp gradient between 60 and 70°C. I get the best results about 60 to 65°C.
Hope that'll help,
Lowering the extension temperature works very well. I haven't tried TMAC.
I tried TMAC to amplify an AT rich gene,
did PCR with 60 mM TMAC, extn at 60 degree,
it didnt work but those reactions without TMAC, extn at 60 degree worked well.