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Primer design for PCR following ChIP - (Dec/23/2008 )

Hi everyone,
I am very new to ChIP (1st time). I've figured out the protocol, but am drawing a blank about designing the primers for the post ChIP PCR analysis. My gene of interest is elk-1 and I am trying to figure out if NFkB (p65) binds to the promoter region of elk-1. There are no publications stating the primer sequence to use. So how do I go about designing the primers (RT-PCR)?
This is what I have done :
I have found the nucleotide sequence of elk-1 using ensemble and identified the 1st exon and 1kb sequence upstream of this. This area contains the TATA box. So do I use this sequence/region to design my primers?? Or is there some other way/easier/logical/scientific of doing this??
Any and all help would be higly appreciated.
Have a great holidays.

-Indigo_nation-

I think you are on the right track. You can read more about how to find promoter sequence of a gene here http://www.protocol-online.org/forums/inde...?showtopic=5063

Beware that Enseml sometimes mis-annotates the first exon. Cross check the promoter sequence you get from Enseml in UCSC genome database (using the blat tool).

Does the binding sites of p65 contain specific sequence motif? If yes, you should search the motif in the promoter sequence and design your PCR primer surrounding the motif.

There are some general rules for designing ChIP primers. Rules for regular PCR primers apply here. ChIP amplicon size should be around 100 bp so that you will get high resolution mapping.

-pcrman-

QUOTE (Indigo_nation @ Dec 23 2008, 11:32 AM)
Hi everyone,
I am very new to ChIP (1st time). I've figured out the protocol, but am drawing a blank about designing the primers for the post ChIP PCR analysis. My gene of interest is elk-1 and I am trying to figure out if NFkB (p65) binds to the promoter region of elk-1. There are no publications stating the primer sequence to use. So how do I go about designing the primers (RT-PCR)?
This is what I have done :
I have found the nucleotide sequence of elk-1 using ensemble and identified the 1st exon and 1kb sequence upstream of this. This area contains the TATA box. So do I use this sequence/region to design my primers?? Or is there some other way/easier/logical/scientific of doing this??
Any and all help would be higly appreciated.
Have a great holidays.


Also, don't forget to design a primer where you don't expect to find p65 binding, for your negative control. Your ChIP data is not meaningful unless you have a background non-binding region to compare to.

-KPDE-

Thanks for replying. I appreciate your help. I'll try your suggestions and get back to you.
A.

-Indigo_nation-