No PCR products with the ChIP DNA - (Jul/31/2005 )

Hi Everyone,

I have been trying to perform ChIP assay, but i cannot get any PCR products in any of my ChIP reactions. My PCR reaction is working as my positive control gives me a single band. I have also tried to increase the amount of DNA, but no luck.
It would be really helpful if I could get some suggestions ..

Thanks

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Have you performed a western blot to show that your Ab is targeting the right protein? also if your IP and input fractions have the protein of interest (by WB)?

How are you isolating your DNA?

Nick

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Yes, I have done WB with the same Ab.
No, I didnt do WB on the IP and input fractions, but will try to do so.
I am using a DNA purification Kit to isolate my DNA.

Thanks for reply,
Pls give me suggestions if u think of any others..will try to do WB.
Thanks once again

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DNA purification using a kit should be no problem at all, although I routinely use the standard phenol-clhoroform method of prepping my DNA.

Also how was your chromatin prepared? was it cross link with formaldehyde? did you decross link after IP?

The Western on your input and IP fractions will tell you how effiecent your IP was with the antibody of interest, the heavy and light chains will light up, so you should get a third band being the protein you are ip'ing.

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How many cycles do you use? Some publications uses 27/28, but some uses 35.

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I think i am losing the DNA onto the columns, coz i cudnt see anything in the Input lane, Yes i did reverse the crosslink, and I have tried using 40 cycles for my PCR reaction.
I am repeating the expt, and will use the traditional purification method and see how things look. Keeping my fingers crossed.
Thank you for your suggestions

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