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I have positive signal in the control without antibody

I have positive signal in the control without antibody - ChIP Chromatin Immunoprecipitation Forum

I have positive signal in the control without antibody - Chromatin Immunoprecipitation Forum. Discuss, chat about, and post questions about the ChIP assay, in the ChIP forum. Includes troubleshooting, protocols, buffer preparation, and more.


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  #1  
Old 03-23-2009, 11:53 AM
Pipette Filler
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Unhappy I have positive signal in the control without antibody



I made Chip DNA in yeast cells and I had a band in negative control, even without usin antibody.
Anyone can help me please?
Thanks.
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Old 04-29-2009, 05:27 PM
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Default Re: I have positive signal in the control without antibody

Lots of possible sources of your problem...

How big is your band in the negative control? Is the ChIP sample one brighter? If so you have an enrichment over background (successful ChIP) and try reducing the PCR cycle number. With some targets the background does come up like this, it's irritating but it's not the end of the world as long as your ChIP band is much brighter.
If your negative control band and ChIP band are about the same that could indicate that your antibody is dud or unsuitable for ChIP, as there is no enrichment over background.
I have always used filtered tips for ChIP to prevent cross-contamination of the negative control immunoprecipitations. If it's your first attempt at ChIP you may find it takes you a few for your technique to get perfected enough to avoid cross-contaminating things.
When manipulating samples I always follow the strict order, MOCK, noAb, ChIP, input to avoid adding anything from tubes containing more DNA to those that should contain very little.
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Old 05-08-2009, 09:47 AM
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Default Re: I have positive signal in the control without antibody

What is the ChIP method that you used (type of beads, blocking, elution) ?
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antibody , control , jeast chip , positive , signal


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