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PCR for ChIP


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3 replies to this topic

#1 RomanA

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Posted 25 November 2004 - 11:41 AM

Hi.
Does anyone knnow how to troublshoot PCR for ChiP. I don't get the product even in the ChIP input although my control PCR using the cloned promoter peace is working. May be the DNa concentration or primer concentration pay a role. I noticed pepole using different primer concentrations...or this is something else...

I'd really appreciate any suggestions.

Roman.

#2 pcrman

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Posted 25 November 2004 - 01:14 PM

Hello Roman,

ChIP PCRs are sometimes tricky. I usually have at least 3 pairs of primers for each locus I want to map and first test those primers using good quality genomic DNA as template. Some primers may work fine with genomic DNA but not with ChIP precipitated DNA. Those pairs just differ slightly in position and length. The primers should be at least 24 bp in size and have over 60% GC.

Also design primers on control genes which are supposed to have the same histone modification as your target gene. Input DNA only serves as a control for starting DNA but not a control for potential DNA loss during the multiple washing steps.

For PCR components, I just stick to my regular PCR formula, and cycle my PCR around 30 times.

You can also check Upstate 2004 catalog for some useful ChIP PCR tips.

Hope that helps.

#3 RomanA

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Posted 26 November 2004 - 10:09 AM

Thanks a lot for your help... well.. at least now I have something to do for my Thanksgiving weekend!

Roman

#4 mikew

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Posted 27 November 2004 - 08:19 PM

Get the primers to work on genomic DNA first. Plasmid conditions don't always carry over to genomic.
Those conditions will work for ChIP.
If they don't you are lossing all of your DNA at some step.
You can even take a small sample of your input and run it on a gel to ensure you actually have DNA.




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