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low yield in library preparation

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#1 guyan



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Posted 06 March 2011 - 01:22 PM

I got a very successful ChIP (DNA sheared to enrichment around 200 bp, more than 2% input at positive controls). Then I used 10 ng ChIPed DNA to make library for sequencing with the NEB kit. But I got a very poor yield (0.4 ng/ul in 30 ul elutes). I repeated twice with the same DNA and the results didn't turn any better. I used the same kit to make another library two months ago and it worked pretty fine. The only difference was that the chromatin was sheared around 200-700 bp (more spread) that time. I always cut gel from 175-600 bp. I really couldn't figure out what's wrong.

#2 Dukey



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Posted 14 April 2011 - 10:05 AM

You basically answered your own question. If you shear too much for ChIP-Seq, you will struggle to get enough DNA following library prep. I personally prefer to shear a little less if sequencing is the end result, say from 900 - 150bp, with the majority below 300 bp. You should see a much better improvement in library yield.

But, 0.4 ng/ul (30 ul) is not terrible and you easily have enough for a sequencing run. The preservation of the specificity in the final library is the most important factor. If you still see the same % input etc in the library then you are good to go.

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