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
- - - - -

CHIP-seq problem with yeast gDNA!!!

chip-seq

  • Please log in to reply
No replies to this topic

#1 lorenzo

lorenzo

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 31 August 2015 - 01:24 PM

hi there,

 

I'm trying to optimize a RAD52  chip seq in S. cerevisiae. 

I'm growing 500ml at 0.5OD of cells, fix with 1% formal for 1h, pellet, bead beat the cells and then sonicate using a covaris (1m volume).(size 100-500bp)

after the whole CHIP protocol, I'm doing a bioanalizer to check the size of the IP and there is almost just a small fat band at 35bp!!

 

what I am doing wrong??!? 

 

doing some optimization of the sonication step I noticed that a huge 35bp band of RNA appears after sonication and I must say that after the IP I didnt RNase treat the sample nor use a qiagen column to purify the IP (I did phenol-chlor and ethanol precipitation.) but even if the small band at the end of the IP protocol is RNA that I can easily remove, I still dont have much DNA in the IP to sequence!! 

 

any suggestions??!?!

 

 

tnx

Attached Files







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

©1999-2013 Protocol Online, All rights reserved.