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ChIP amplification problems


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#1 Dave_Kub_11

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Posted 23 November 2011 - 05:01 AM

Hi there,

I have a problem with amplifying my ChIPs using qPCR. I originally used diagnostic PCR and was only able to amplify up my Histone H3 positive control. Since swithcing to qPCR I was able to get results with one of my experiments and can view differences in binding by gel electrophoresis.

However, with the next set of experiments, I've had a strong band pop up in my IgG lane with 2 different primer sets that is the same intensity as all the other bands there. I've tried fresh SYBR green, primer dilutions, everything and still had this problem. For another primer set, I've then tried stopping the reaction before the curves level off so I can measure the differences, but alas I had lots of smearing in all my lanes. I've also tried diluting down the DNA 10 and 100-fold and I still got a band in my IgG and nothing in my Histone with the same primer set.

I am ChIPPing with IgG, Histone H3, Sprouty2 and HIF1 alpha on various regions of the VEGFA promoter.

I followed the Millipore protocol and used a nucleospin kit to extract the DNA.

Anyone have suggestions as to my qPCR woes?

I would really appreciate the help,

Thanks,

Dave

#2 chabraha

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Posted 29 November 2011 - 03:54 PM

try pre-clearing your samples with beads alone, also do PCR at a negative control region (this control is more important than the IgG control).......and I'm not sure what your referring to by qPCR, but you should do real-time PCR. Different experiments often have variation in the amount of chromatin you recover......just because you saw differences in the band intensities doesn't mean you are viewing the PCR results in the linear range of amplification.
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