I used 4 month old stored DNA @ -20 deg C. I got good PCR bands but not good result for pyrosequencing. Would it help if I store @ - 80 deg C and make aliquots of that?
hi Sujata,
It might depend on the manufacturer of the kit. Qiagen for ex. states that with their epitect kit, bis conv. DNA remains 80% intact after 3 years at -20°C. I´m using the Zymo kit for conversion. I just used 18 months old bc DNA for analysis, and it worked great.
If your DNA is degraded at -20°C within 4 months it will probably also do at -80°C. The problem might lay somewhere else...
Have you checked new samples with the same pcr primers and same pyro sequencing primer?
Hi Olivier,
Thanks a lot for your answer!!
I am also using EZ gold methylation kit from Zymoresearch. I checked with other samples and the primers are working well. What may be the possible reason for not getting good quality pyrogram with desired peak intensity??
Hi,
I remember that a few years ago, we had similar problems with bisulfite treated DNA prepared with a Zymo kit. Methylation specific PCRs were absolutely fine, but the pyrosequencing was of poor quality. Changing to the Qiagen bisulfite kit solved the problem.
I have also used several year-old bisulfite treated DNA for MSP (not pyrosequencing), and that was fine too
- Badges
- Science method