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.


problems with PCR

problems with PCR - ChIP Chromatin Immunoprecipitation Forum

problems with PCR - 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 11-11-2008, 02:08 PM
Pipette Filler
Points: 2,681, Level: 33 Points: 2,681, Level: 33 Points: 2,681, Level: 33
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2008
Posts: 24
Thanks: 0
Thanked 0 Times in 0 Posts
Default problems with PCR



Hi,
I have some questions concerning my ChIPs.
1. Why don`t some primer work in the eluates, but do in the input?
2. Why are there often some bands (unwished) in the negative conrol that aren`t in the input, also there is more product in total?
3. Why do some primer that work well on genomic DNA, just give a smear in the ChIP?
Thanks a lot in advance...
Reply With Quote
  #2  
Old 04-29-2009, 06:02 PM
Pipette Filler
Points: 74, Level: 1 Points: 74, Level: 1 Points: 74, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2009
Posts: 8
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: problems with PCR

1. The input is the positive control for your reaction and should be taken from the same material as you intend to ChIP. If primers produce a band in the input but not in the ChIP eludate then this region is not enriched and your target protein does not associate with it. This of course assumes you have a suitable positive control showing that another region can be immunoprecipitated from the sample and thus there is nothing wrong with the sample or the protocol.

2. This suggests your primers might recognize multiple sequences under the PCR conditions you've used. Try increasing the annealing temperature or following other aspects of a PCR optimisation strategy (there are lots online) to increase the specificity of the process. BLAST your primer sequences to the genome of your species of choice to check for other sequences that could bind your primer. If the product you describe is smaller than 100bp then it could be primer dimer forming in the absence of any sequences to bind to in the sample and I wouldn't worry too much about it unless you plan to do quantitative PCR with intercalating flurophores.

3. ChIP DNA is genomic DNA so there is no reason that it shouldn't work unless the DNA's in question are from different species. But ChIP DNA is fragmented genomic DNA of course. Are you trying to amplify too much sequence? PCR becomes difficult with primers amplifying more than 250-300bp on ChIP because the polymerase falls off the fragmented ends before it completes the amplification round. This would result in a smear on the gel, could this be your problem? Keep your amplified region small.
Reply With Quote
Reply

Tags
pcr , problems


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
problems with growing healthy Arabidopsis plants Monika Kuzma Arabidopsis and Plant Biology 1 02-19-2007 03:55 PM
Problems with growing healthy Arabidopsis plants - light intesity? Karl Lundy Arabidopsis and Plant Biology 0 02-07-2007 05:11 PM
[ANN] xmds-1.3-4 released! xmds solves complex problems simply and quickly Paul Cochrane Forum Physik 0 06-21-2004 01:34 AM
[ANN] xmds-1.3-4 released! xmds solves complex problems simply and quickly Paul Cochrane Forum Chemie 0 06-21-2004 01:29 AM


All times are GMT. The time now is 02:33 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 0.12209 seconds with 16 queries