SEQanswers

Go Back   SEQanswers > Applications Forums > Epigenetics



Similar Threads
Thread Thread Starter Forum Replies Last Post
ChIP-Seq: knockout as control? Combo Bioinformatics 4 11-03-2011 05:44 PM
ChIp seq Library control frigo Illumina/Solexa 0 10-28-2011 05:23 AM
ChIP-seq DNA concentration jazz Illumina/Solexa 1 10-07-2011 07:16 AM
Anyone use low concentration for ChIP-seq prep? Audry888 Sample Prep / Library Generation 4 05-28-2010 11:37 AM
concentration of my starting material? seqgirl123 Illumina/Solexa 6 07-28-2009 06:19 PM

Reply
 
Thread Tools
Old 12-07-2011, 06:52 PM   #1
mnandita
Junior Member
 
Location: Bangalore, India

Join Date: Dec 2011
Posts: 7
Default Starting concentration of sample/control in ChIP Seq

Given the little amounts of DNA obtained after ChIP, are analysis algorithms equipped to normalize data across input/mock/test ips if different amounts of starting material were used in each case to make the libraries?

So if i have 10 ng of IP test, but 40 ng of input and 100 ng of mock, should I necessarily make all libraries from 10 ng only, or can I take all the DNA I have for each of the experiments?
mnandita is offline   Reply With Quote
Old 12-15-2011, 07:33 AM   #2
ETHANol
Senior Member
 
Location: Western Australia

Join Date: Feb 2010
Posts: 308
Default

You should absolutely use the smallest amount of DNA from your samples in all your libraries. In your example that would be 10 ng. The main reason is because PCR induces biases with each cycle (exponentially I believe), so if you are amplifying one sample less then other, the PCR bias won't be transferred equally between them.

More simply, samples should be treated equally, to do that you need to use the same amount of starting material.

One other thing, mock IP isn't really a good control. Just use input and your ChIP.
__________________
--------------
Ethan
ETHANol is offline   Reply With Quote
Old 12-15-2011, 09:02 AM   #3
mnandita
Junior Member
 
Location: Bangalore, India

Join Date: Dec 2011
Posts: 7
Default

Thanks Ethan. I agree with that (reg. starting with same amounts).

As for the mock control- if your Ab is not very good, and there is sufficient amount of non-specific DNA pull down that does not show up in the input, how do you control for it without a mock? I do not think the mock is dispensable, except in cases where you have a very specific Ab.

Last edited by mnandita; 12-15-2011 at 09:04 AM.
mnandita is offline   Reply With Quote
Reply

Thread Tools

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




All times are GMT -8. The time now is 04:04 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO