ChIP: I get smear after PCR? - (Jan/17/2007 )

Hi there,
I have a great problem.
I am performing ChIP. The first time I did the experiment I got good results meaning that there was a positive clear signal with the Ctrl ab and no signal with the negative ctrls. Fine.
Then I repeated and all I got from PCR was smear from the input, from the IP., ...The product is supposed to be around 800 bp.
Then I started all again and again I got smear from all of the samples.
I have attached two pictures( ONe showing my Chromatin after sonication; the other PCR).
HAs anybody seen something like that??
or have an idea??
I am trying to find the reason for long time and peopole do not have much idea why it might be so.
I would be very happy if you could give me an idea
Thanks a billion...

-clementine-

QUOTE (clementine @ Jan, 12:39 PM)

Hi Clementine,
Looks like you have too much template. The product is high mw dna. How about titrating your template. I was able to amplify from one hundredth of a dilution of Iped dna. Second, you may titrate Mg. Try lower concn. good luck
C

-Chipologist-

Hello,

I think 800 bp is too long for ChIP PCR. By saying that I mean ChIP is designed to be able to achieve high resolution than 800 bp, do not mean you cannot amplify fragment > 800 bp, although the smaller the easier. Regardless, I would suggest that you redesign 2-3 pairs of primers for every region to be covered with a PCR product of 150-200 bp and rerun the PCR using less template. Hope that works.

-pcrman-