Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Epigenetics Forum: DNA Methylation, Histone and Chromatin Study > ChIP Chromatin Immunoprecipitation Forum
Register Search Today's Posts Mark Forums Read

ChIP Chromatin Immunoprecipitation Forum Chromatin Immunoprecipitation Forum. Discuss, chat about, and post questions about the ChIP assay, in the ChIP forum. Includes troubleshooting, protocols, buffer preparation, and more.


ChIP Assay Sonication Problems

ChIP Assay Sonication Problems - ChIP Chromatin Immunoprecipitation Forum

ChIP Assay Sonication Problems - Chromatin Immunoprecipitation Forum. Discuss, chat about, and post questions about the ChIP assay, in the ChIP forum. Includes troubleshooting, protocols, buffer preparation, and more.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-11-2008, 11:14 PM
Pipette Filler
Points: 2,365, Level: 31 Points: 2,365, Level: 31 Points: 2,365, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2006
Posts: 21
Thanks: 0
Thanked 0 Times in 0 Posts
Angry ChIP Assay Sonication Problems



Hello all, im getting problems with my ChiP sonication steps. I follow the steps from the Santa Cruz kit. Im using all the reagents according to the manufacturers instructions.

The problem is that when I sonicate my 293T cell nuclear extracts, and then I reverse cross-link, and then phenol extract my DNA, I get only a DNA that smears.
Can someone hand me some suggestions please?
thanks
Reply With Quote
  #2  
Old 09-15-2008, 09:38 PM
Pipette Filler
Points: 285, Level: 5 Points: 285, Level: 5 Points: 285, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2008
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: ChIP Assay Sonication Problems

You should get a smear, as the sonication is random. The smear should ideally be between 150-500 bp though may extend to 1-2kb. The upper limit of the smear is the resolution size of your target sequence.
Reply With Quote
  #3  
Old 02-17-2009, 10:55 AM
Pipette Filler
Points: 2, Level: 1 Points: 2, Level: 1 Points: 2, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Thumbs down Re: ChIP Assay Sonication Problems

Hi:
I have tried to do the ChIP assay in L6 cells. I use lysis buffer that contains 1%SDS, 10mM EDTA, 50 mM tris-HCl pH 8.1. I add freshly protease inhibitors and PMSF. I sonicated 1x15 sec, 3x 10sec and after an hour in 65 degrees with NaCl, SDS and proteinase K I saw 2 bands one around 650kb abd one around 1000kb. I didn't get the regular smear I used to.
Does anyone has any idear why???
Thank you, Shiri
Reply With Quote
  #4  
Old 02-17-2009, 02:12 PM
NATHANALANPATRICKROGERS's Avatar
Summer Student
Points: 306, Level: 6 Points: 306, Level: 6 Points: 306, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Maroochydore
Posts: 73
Thanks: 4
Thanked 13 Times in 11 Posts
Default Re: ChIP Assay Sonication Problems

The primary purpose of the extractions are to mobilise the DNA and other cellular constituents. The size and quality of your DNA [as verified by gel analysis] depends on both how well you perform the extraction and the extraction technique itself.

Think of extraction tools like sonication as a fork that stirs a hot bowl of noodles. Before the noodle cake is stirred for the first time, all the noodles are roughly the same length but they are interwoven. As you stir, the noodles break apart into smaller pieces but also get softer. This effect is likely to explain why you sometimes encounter smears, other times bands and inevitably nothing at all.
Reply With Quote
Reply

Tags
assay , chip , problems , sonication


« - | Strain specificity »
Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
ChIP Assay DNA Extraction Kit? Qiang ChIP Chromatin Immunoprecipitation Forum 3 05-08-2009 09:37 AM
Bradford assay - protein quantification using BSA Standard. but i encounter problems foRk Protocols and Methods Forum 2 10-22-2008 02:17 PM
ChIP assay fatima Molecular Biology Techniques 0 03-07-2008 11:26 PM
ChIP assay for the study of methylation and chromatin remodeling moleculardude Epigenetics Forum: DNA Methylation, Histone and Chromatin Study 5 11-12-2006 07:02 AM


All times are GMT. The time now is 09:52 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12864 seconds with 16 queries