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Ampification in no-antibody control of ChIP assay


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5 replies to this topic

#1 seenew

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Posted 27 December 2004 - 12:45 AM

Hi,

Merry Chrismtas and happy New year.

I am working on Chromatin immunoprecipitation now. I am so upset that my no antibody control also has PCR band. Did anybody meet this kind of problem? What should I do? By the way, I use the ChIP kit from Upstate. I did no template control when I did PCR. There is no band. So it is not because of PCR contamination. Thanks

#2 mikew

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Posted 27 December 2004 - 12:05 PM

Difficult to know for sure, especially if you have a good negative control for PCR contamination. My suggestions would be to preclear for a longer period of time, wash for a longer period and possibly do extra washes.

#3 seenew

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Posted 28 December 2004 - 09:07 PM

Thanks a lot. I will try.

#4 luminb

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Posted 29 December 2004 - 11:53 AM

in addition to prolong the preclear and washing cycles, also be aware of your primer specificity and pcr cycles. there will be some non-specific dna stuck on the beads for sure, but usually PCR using your primers will not amplify it if your primers and pcr program are good. that is why i always use primers with tm around 80 degree. why don't you check your primer Tm and raise the annealing temperature?

#5 pcrman

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Posted 29 December 2004 - 03:43 PM

I once talked to Upstate technique support about non-specific chipping. Here are their recommendations:

As I had mentioned, you may be experiencing non-specific binding of proteins to the salmon sperm/protein A agarose. To pre-block the agarose, please perform the following:

Incubate/wash the Salmon Sperm DNA Agarose before using it. It can be blocked in 1-5% BSA and Chip dilution buffer to "block" the agarose matrix (you can incubate for 30 minutes mixing at room temperature). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernantent. Wash once in ChIP assay buffer and continue with the assay as normal.

BTW, I usually get no amplifications in no-antibody controls even without prewahsing.

Hope that helps.

#6 seenew

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Posted 04 January 2005 - 08:53 PM

Thanks all of you. I used 1 hour for the preclear step( the kit suggests 30min). Sometimes the no antibody control had no band, sometimes had. The size of the non-specific band is the same as my target fragment. So it still will be there even I change my primers. Maybe I should block the agaose mix first.

Edited by seenew, 04 January 2005 - 08:58 PM.





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