Tricks to amplify 4kb targets from GC rich genomic DNA - (Nov/27/2008 )
Problems with amplification of 4kb pcr products from GC content rich genomic DNA. I have tried several approaches by adding DMSO, longer initial denaturation time and extension time but still no bands are yielded. I am now using Sigma AccuTaq and below are the temperature programing I have set for the experiment:
Initial Denaturation: 95oC for 3 mins
30 cycles of
55-58oC, 1min (the Tm for my primers are 62-64oC)
Final extention: 72oC, 5min
Are there any other tricks that I have missed out? This is indeed frustrating!
Add 5% betaine, it works much better than DMSO. What enzyme are you using? Taq will probably not work for this. Phusion would be my first choice. You might want to redesign the primers -- not all of them work, and it's cheap insurance. How certain are you of the genomic sequence you are binding to? Might there be an intron?
Yes, what primers are you using?
I used an iDNA master mix with 1.5ul 10pmol primers each in 50ul reaction. Cycling:
1. 95 C 2min
2. 94C 20sec
3. annealing 1min
4. 68C 3min
rpt 2-4 39x
5. 68C 10min
But my primers were T3T7 primers.
If you use normal buffers increase the dNTPs and primers a bit and do a 50ul reaction volume.
Hallo, in portfolio of the Ecoli.sro there is special polymerase for GC rich amplification www.ecoli.sk
they provide products from the Genecraft
I'm with phage434: 5% Betaine is the way to go, and try Phusion polymerase.