Bisulphite conversion - (Jun/19/2013 )

Hi all,

I think I have done a silly mistake and I need an immediate help.

During Bisulphite conversion of DNA using EZ DNA Methylation-Gold™ Kit, my PCR did not worked for these conditions.

1. 98°C for 10 minutes
2. 64°C for 2.5 hours
3. 4°C storage up to 20 hours

I did not notice and just followed the further steps. I just collected my samples from thermal cycler.
Can I use this eluted DNA for retreatment with bisulphite and follw the standard protocol using above kit.

Thanks for the help.

Your PCR did not work does not necessarily mean the problem is at the conversion step. Many factors apart from DNA modification could lead to failure in PCR and in most case, the problem is at PCR: primer design, PCR condition, etc. What type of PCR are you doing and what are your PCR conditions?

I think what he meant was that the cycler, which was supposed to run the conversion reaction for 2.5 hours at 64 degrees failed to start. I suppose the DNA is still present, but I certainly would go back and restart the experiment rather than trying to rescue a potentially trashed sample.

You got me right phage434. I think you are right. I should go with the new experiment rather to be confuse with the results and wasting of chemicals.

Thanks

Epi_Man on Wed Jun 19 15:25:09 2013 said:

Hi Epi-Man

I'm also working with the same kit, and unfortunately I'm also really confused because my PCR did not worked. I have some doubts about the amount of the input DNA. No other thing could be went wrong....
please let me know if you've solved the problem

Thanks

A much more likely problem is a bad choice of primers. Check your primer design carefully.