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
- - - - -

questions about shearing chromatin analysis


  • Please log in to reply
3 replies to this topic

#1 echolee76

echolee76

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 19 April 2012 - 07:11 AM

Hi all,

I did the chromatin shearing with Biruptor.( condition is Power "high". 10 times for 30 seconds at 30 second intervals.) The gel pic looks like a lit bit strange. I could not find the genomic DNA band on unsheared samples. Who could you give me some idea.
In the gel pic, I have 5 different concentration cell samples. The sample marked U is that unsheared sample and marked S is that sheared samples. The unsheared samples i took them before put in Biruptor and directly load on the gel without add any RNAse and incubation. I need help to continue. If i have problem with cross-link or lysis buffer.

The lysis buffer recepie is
Lysis Buffer fresh reagent [final] Tris–HCl, pH 8.0 50 mM EDTA 10 mM SDS 1% (wt/vol) protease inhibitor mix 1x PMSF 1mM Na-butyrate 20mM


Many thanks!

Ting

Attached Thumbnails

  • 17.04.2012.png


#2 chabraha

chabraha

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
2
Neutral

Posted 23 April 2012 - 10:12 AM

since you didnt reverse the crosslinked chromatin you can not expect your unsheard sample to run into the gel............my guess is that what you are primarily looking at is RNA in all of your lanes. You need to reverse the crosslinks and RNAse treat your samples to get any meaningful information on whether your shearing conditions are good or not..............your lysis buffer looks fine to me.
Treasure Chest Wizardry

#3 echolee76

echolee76

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 03 May 2012 - 07:59 AM

Hi chabraha,

I doubt my lysis step did not work and the shearing was ok.So I have repeat this lysis procedure again. This time i add the 20 gauge needle treatment and reverse cross-linking step. The result see the attachment. This time i could find the genomic DNA band in the unsheared lysis samples. So i think I could continue to next step, do you think so. Thanks:)

Attached Thumbnails

  • 23.04.2012 ChIP Lysis test.jpg


#4 chabraha

chabraha

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
2
Neutral

Posted 03 May 2012 - 08:10 AM

Sure.......I run my samples 6 times through a 20g needle and it definitley helps crack the nuclei and slightly shear the DNA....just be sure to RNAse your sample before you load it onto your gel...........otherwise you can't be sure whether you are looking at RNA or DNA.
Treasure Chest Wizardry




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

©1999-2013 Protocol Online, All rights reserved.