Hi. I have problems to amplify a fragment by PCR which has a high GC content. I've tried with DMSO at 3% and 5%... but nothing. I've tried modifying the conditions like temperatures and times...but nothing. Could anybody help me? Thank you very much.
I agree with the above suggestions while finding the Roche long PCR kit very good.
If all else fails there is an interesting paper on the use of a mutated taq polymerase in strand displacement PCR to get round the high GC content problem
Ref in Biotechniques
A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification
Konstantin B. Ignatov1,2, Ekaterina V. Barsova3,4, Arkady F. Fradkov3,4, Konstantin A. Blagodatskikh2,5, Tatiana V. Kramarova6, and Vladimir M. Kramarov1,2
Hello Favio,
This may not be helpful but, Invitrogen's 10x PCR Enhancer solution and NEB's GC Buffer (for Phusion polymerase) are for such purposes. Maybe if you can find what's in them or what is different about them you can figure it out.
Good luck!
Try supplementing the reaction with 1M betaine. This is supposed to eliminate base pair composition differences in PCR reactions.
I agree with the above suggestions while finding the Roche long PCR kit very good.
If all else fails there is an interesting paper on the use of a mutated taq polymerase in strand displacement PCR to get round the high GC content problem
Ref in Biotechniques
A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification
Konstantin B. Ignatov1,2, Ekaterina V. Barsova3,4, Arkady F. Fradkov3,4, Konstantin A. Blagodatskikh2,5, Tatiana V. Kramarova6, and Vladimir M. Kramarov1,2
As DMSO activity is similar as GC rich solution, If you got GC rich try this one out and increase your annealing temperation if required.
Hi Favio,
I have found that not only does 1M betaine work great in high GC PCR, but if you want full length or fairly long transcripts, you should also supplement your RT reaction when you make your cDNA. The betaine is supposed to relax secondary structure, and higher temp also helps (use something like Thermoscript from Invitrogen). Betaine (5M PCR reagent) is inexpensive from Sigma.
Favio,
You can try to go up to 10%DMSO, Betaine (1M final concentration) also can work. Yo can also try to increase the denaturing step temperature to 97ºC in you PCR program.
Try changing the Mg+ concentration.. You can also mix taq and phusion enzymes in different ratio and try..
I don't have DMSO for PCR in the lab but have Analytical reagent (AR) with 99.7% purity. Is it possible to use it for PCR?
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