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Old 09-19-2008, 12:56 AM   #1
athos
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Default ChIP seq sample prep for Illumina

Hi Everyone, I'm hoping someone can give some advice on ChIP enriched DNA. Does anyone try and quantify this by nanodrop/picogreen before starting the Illumina sample prep protocol (the protocol recommends starting with approximately 10ng DNA)? We have had difficulty in quantifying samples reliably. Is anyone doing ChIP seq regularly that can help?
Thanks.

EDIT : Sorry, now posted in Illumina section!

Last edited by athos; 09-19-2008 at 01:11 AM.
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Old 11-20-2008, 06:34 PM   #2
tomv
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It's possible but not practical to quantify your IP DNA. To do it you need to either resuspend it in a small volume (10-15uL) and use a nanodrop or use a SYBR assay. In practice we only do this occasionally, such as when we are testing a new antibody or if we suspect a problem. Beyond that we just use the yield after LM-PCR as our first QC checkpoint.

I wouldnt' worry about getting exactly 10ng input. What is more important is to empirically optimize the concentration of adaptor that works in your hands. The illumina protocol calls for something like a 1:10 ratio of insert to adaptor but there is a wide range of IP yields dependent on protocol, antibody and phase of the moon so you really need to do the titration yourself. We find for ChIP-seq that something between a 10 and 100 fold dilution of the adators in the genomic DNA kit works for us.
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Old 12-01-2008, 10:01 PM   #3
weiweideng
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Because of the lower concentration of the ChIP DNA samples, it is not accurate to check it by nanodrop, it's better to use picoGreen stain.
Although the Illumina's protocol recommend 10ng is enough, many companies could not realize this for library preparation. And you also could not get a good result to use only 10ng. It's better to use more than 100ng. Gook luck!
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