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Old 07-07-2011, 09:08 PM   #1
shurjo
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Location: Rockville, MD

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Default removal of primer dimers.

I'm trying to tweak the Illumina RNA-Seq library prep to convert a library of blunt-ended PCR amplicons (sized between 200-600bp) into sequencer-ready form. The starting material is in the low nanograms (<10). I added A overhangs, ligated the PE adapters (using 0.1ul), size selected 250-650bp on a gel and did 15 cycles of PCR with 1.0ul of each primer. The results from running the final product on a BioAnalyzer DNA HS chip are shown in the attached picture. Does anyone have suggestions for getting rid of the two peaks below 150bp (I'm guessing these are adapter or primer concatemers)? I am thinking of SPRI beads or a Pippin Prep run. The obvious choice would be to repeat the whole experiment with lesser primer in the final PCR, but I have limited RNA availability and if this library can be rescued that would really make my day.

Thanks,

Shurjo
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Last edited by shurjo; 07-07-2011 at 09:14 PM.
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Old 07-07-2011, 09:29 PM   #2
ECO
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Hey shurjo,

It's not my important experiment, so I can say the following with extreme confidence ( )..., but a 0.8x AMPure should remove most of that. If you're feeling particularly daring, 0.6x could be better. Either way I'd save the supernatants in case you don't get the purification you need (just add more AMPure to it to >1.8x final to recover everything >~100)

Good luck!
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Old 07-08-2011, 02:12 PM   #3
shurjo
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Thanks ECO! Will try this out and post again once I have the results.
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