Optimising bisulphite PCR primers - (May/16/2008 )
I need some help in optimising my bisulphite PCR primers. I have six sets of primers covering my area of interest. Four of these work. However I have two sets of primers which will not work. I have gone as low as 48C but still have not had any product. As the converted DNA now has long stretches of poly A and Poly T could it be possible that I am getting hairpin loops forming? - I could perhaps use DMSO? Or should I increase the number of cycles from 30? Any advice is very much appreciated. Xmun
1. Design new primers, while you troubleshoot this.
2. I hope you are using hotstart taq.
3. Have you tried PCR buffer containing 0.1% w/v Tween-20?
The best way is to design new primers.
Both yourself and larryking advise designing new primers. I am a complete beginner at bisulphite PCR so please excuse what may be a stupid question: I have used both Methprimer and Methyl Primer Express to design the primers, and chose the ones that seemed best. If these are theoretically the optimum primers, why should suboptimal primers work where these did not?
However I see from the threads that everyone does two rounds of PCR and uses nested PCRs. I have not, it seems I did not read up enough. So it may be simply that I need to run a second PCR or maybe design first round primers. This raises another question: As all the protocols advise keeping product size to under 400bp, and suitable primer binding sites are very restricted, what is the maximum product length advisable for the first round of PCR?
Also: is the funtion of the Tween-20 to prevent hairpin loops? I have already tried DMSO.
Thanks for the patience
Try lowering your extension temperature to 65 degrees. High AT regions will not extend at normal PCR extension temperatures.