Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Bisulphite conversion

bisulfite conversion PCR

  • Please log in to reply
5 replies to this topic

#1 Epi_Man

Epi_Man

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 19 June 2013 - 07:25 AM

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.


#2 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
68
Excellent

Posted 19 June 2013 - 08:58 AM

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?

#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,470 posts
247
Excellent

Posted 19 June 2013 - 09:29 AM

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.

#4 Epi_Man

Epi_Man

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 20 June 2013 - 03:04 AM

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

#5 nazanin kia

nazanin kia

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 26 June 2013 - 11:07 PM

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.


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

#6 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,470 posts
247
Excellent

Posted 27 June 2013 - 05:42 AM

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





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.