PCR problem - PCR did not work (Oct/30/2008 )

Hi
I am working on PCR of 1.7 Kb cDNA fragnment. It has high repeat of nucleotides. I tried with touch down method, Nested PCr and PCR using all the primers combinations. Positive and negative controls are working well. Problem is I can not amplify the band, some time I can but when I sequence, it appears false. I tried DNA premix, all kind of polymerases available in market... Any body has any trick to recover the problem??

Junior Obama, S. Korea

Are you sure your cDNA is ok?

Yes I checked the cDNA loading on gel. It has no problem.

Hello

You can try betaine

I used the concentration of betaine as described in paper and temperature 53 to annealing and works greate.


Betaine improves the PCR amplification of GC-rich DNA sequences.
Henke W, Herdel K, Jung K, Schnorr D, Loening SA.
Nucleic Acids Res. 1997 Oct 1;25(19):3957-8.


Good luck!

do you have your primer seq only once in your template (as you are talking of repeats in you seq;); maybe you are generating artefacts because your conditions are not strigent enough?
Or are you getting more than one band?

Maybe a gradient PCR helps?

I am facing similar problems time and again (although with genomic DNA); and usually the only thing that really helps is to try different annealing/elongation times, touchdownPCR, gradientPCR different enzymes and different additives (like already suggested betaine or DMSO, BSA, etc.); different number of cycles etc. until you get fine results.....

Thanks for your suggestions. I used all of the methods suggested above except betaine. I will try it and let you know the progress. My PCR problem is most of the time I do not get band, if I get band it appears false sequence and even result is not consistent. Actually, my former labmate found the mutated region by fine mapping in rice genome. He bought the cDNA sequence of that region from somewhere ealse. He found that there is splicing error and in the begining of splicing point there is a point mutation from A to G. Just he left. Another guy tried for about a year but failed to clone. Now I am taking it.