DNA bisulphited Amplification
Posted 08 July 2010 - 02:04 PM
Then, we tried to digest or not the DNA, used a different Sodium Bisulphite, changed the PCR's conditions, but nothing happened!. Moreover, the DNA and Magnesium Chloride concentration were modified, and the primers and enzymes were changed. Finally, a nested PCR (using different cycling programs) was tried. But there is no band!
Any help is welcome,
Posted 08 July 2010 - 03:29 PM
Posted 08 July 2010 - 04:42 PM
post the sequences too
All comments and communication are my own personal ones, and are not tied to any of my affiliations.
Posted 09 July 2010 - 05:26 AM
Yes, Initially the primers were working! But now they began not to walk. We checked the sequences a lot of times!
With the traditional PCR (three steps: denaturalization, annealing and extension) not band was detected, therefore we designed one of only two steps: denaturalization to 80°C 15 seconds and annealing + extension to 60 ° C 60 seconds. Every was very well with this cycle, but only hard a time ...! We know that the DNA is unstable, so inclusive we have proved it with DNA newly bisulphited.
With regard to the cut, we follow the instructions of the specification sheet of the enzima, know that the domestic purification might be a point in against.
Forgive my Englishman's mistakes! And Thanks again!!