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What in ChIP samples inhibit PCR? - (Oct/18/2007 )

Hello, everyone!

I am trying to work out ChIP using mice liver tissue. I tried to do a concentration curve using total Input smaple. I used 5ul, 2ul, 0.5ul, and 0.2ul of Input sample. To my surprise, everytime, 5ul well PCR did not work. So I guess that something in the sample may inhibited PCR. I use stratagene 1-step brilliant Q-RT-PCR kit except RTase, it means without RT step. After reverse cross-linking, I used 1ul of 10mg/ml proteinase K 1 hour incubating samples at 45 degree.Qiagen PCR purification kit was used to purify DNA and 10mM Tris-HCL to elute the DNA. I tried to boiled the sample for 10 minutes to inactivate the proteinase K, but this did not help. I appreciate any help or any suggestion.



In my experience, I decrosslink overnight and do not use proteinase K at all. it works.
Maybe in 5ul input you have too much template that it inhibits PCR? That's also possible.


Thanks for your reply, COCOMILK! I will try not add proteinase K next time. 2ul of total Input C(T) is about 23-25, this is why I tried to use more sample.
Have a good weekend. rolleyes.gif