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Old 02-06-2009, 12:45 PM   #1
jbot
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Default Amount of ChIP DNA required for Solexa seqeuncing

Hi-
Illumina recommends using between 10-50 ng of ChIP DNA for a sample prep. Does anyone have experience with using less than 10 ng? I tried using about 2 ng (it's all that I had), figuring the worst that could happen would be to get fewer reads than usual, but I ended up getting basically no reads at all. I calculated the amount of PCR-enriched DNA to load onto the flow cell based on the A280 reading. I couldn't see the PCR-enriched sample on the Bioanalyzer, but I figured that this wasn't a big deal, as another sample that gave excellent sequencing results was only barely detectable on the Bioanalyzer.
Does anyone know if there is a general problem with using less than 10 ng starting material?

Thanks!
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Old 05-21-2009, 05:03 PM   #2
DSB
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If you are using the Nanodrop for measuring quantity of DNA, this can be out by an order of magnitude or more. I don't lnow if this generally applies to other A280 readings at low concentrations. The recommendation is to use the Picogreen assay or a RT-PCR of properly adapted sequences for all quantitative measurements .
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Old 05-24-2009, 05:36 PM   #3
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I agree that the Nanodrop readings can be way off. But does anyone know whether ChIP-Seq generally works ok if one uses less than 10 ng of DNA to start off with? I quantify the concentration of ChIP DNA using a PicoGreen assay and have had no trouble when using more than 10 ng to prepare the sequencing library -- but what happens if one uses less?
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Old 01-13-2010, 02:59 PM   #4
xenia.zhang
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I think it's problematic if you can't detect any PCR enriched DNA on bioanalyzer,
however, 1ng to 10ng starting material for lib. construction worked.
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