Quality of Bisulfite Converted DNA
Posted 04 February 2013 - 08:02 AM
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?
Posted 04 February 2013 - 10:12 AM
Posted 05 February 2013 - 01:32 AM
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.