Quality of Bisulfite Converted DNA - (Feb/04/2013 )
I'm having some problems with my Bisulfite PCR. I cannot amplify the target fragment.
At first I thought it could the primers, but I went trough all of them to see if I had done something wrong. All the parameters look good!
I designed them using Meth primer software.
So, I'm thinking that it could be the quality of the converted-DNA. I converted it using the QIAGEN EpiTect kit.
I have quantified it using Nanodrop and agaroses gel. It looks fine, but on Nanodrop you can see a peak at 230nm (higher than on 260nm).
Does anyone know how to be sure of the DNA quality?
It is very hard to check DNA quality after bisulfite treatment because the DNA is tiny in amount and has been degraded considerably. Measuring concentration on Nanodrop won't give you much information. If you get high reading on 230nm, it could be that the sample has high salt concentration. Because for bisulfite PCR, many factors not just template could contribute to PCR failure such as primer design, PCR conditions, you need to troubleshoot all possible causes. Can you post your PCR conditions here?
Thank you for the reply!
I'm using the Pfu Turbo Cx hotstart polymerase. It's a proofreading polymerase and it also allows to read uracil throughout the DNA.
The final concentration of the reagents are:
250uM of dTNPS
0,5uM of both primers
1U of polymerase
60ng of Converted DNA
The PCR steps are: 95C for 3 minutes; 95C for 30 seconds, 60-45C for 30 seconds (I made a gradient), 72C for 1 minute (25x this cycle); 72C for 10 minutes.
I also saw that, when I've designed the primers, the Meth primer software gave the Tm of the primers around 58C, but when I received the primers from the company, the Tm was around 43-45C.
When I use any available Tm calculator online, it gives me the Tm around 58C.