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
- - - - -

Sonication Help!

Sonication RNaseA Proteinase K Reverse Cross-link

  • Please log in to reply
3 replies to this topic

#1 j47027

j47027

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 19 April 2013 - 08:29 AM

Hi,

I am assaying sonication conditions at the moment and getting confused with the order of steps.

When I shear my chromatin what do I do next.

RNaseA treatment - 2 hours
Proteinase K - overnight*
Reverse Crosslink overnight*
Phenol Chloroform - Next day

Is that the correct order of steps?

I don't have to use Proteinase K either..correct? and *If I choose to use Proteinase K I can reverse crosslink along with Proteinase K digestion..yea??

Many thanks

Joe

#2 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
67
Excellent

Posted 19 April 2013 - 11:30 PM

Are you doing sonication for ChIP assay? If yes, the next step is incubation with a desired antibody, then washes, elution, reverse cross-linking, digestion ...

#3 j47027

j47027

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 20 April 2013 - 08:30 AM

Are you doing sonication for ChIP assay? If yes, the next step is incubation with a desired antibody, then washes, elution, reverse cross-linking, digestion ...


Yea I am..But I am only at the stage of sonication..I want to analyse the DNA size range before I go through with the ChIP..

#4 Vassil

Vassil

    member

  • Active Members
  • Pip
  • 16 posts
0
Neutral

Posted 30 April 2013 - 05:19 PM

Hi Joe,

If you only want to check the shearing of your DNA, the steps are correct.

I usually incubate the samples with RNase for 30min at 37oC and with Proteinase K for 30min at 37oC or 65oC, but overnight also works. One thing that you may try is to boil the samples (or put at 100oC) for 30min and then do RNase/PRotK treatment for fast decrosslinking.

You may also try the QiaQuick PCR purification kit or MinElute PCR purification kit from Qiagen to get your DNA. There is also a fairly good product from Feldan.

Good Luck!





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

©1999-2013 Protocol Online, All rights reserved.