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-   -   Wang, 'Low Cost Library Construction...': Low Concentration (http://seqanswers.com/forums/showthread.php?t=40859)

MAdkisson 02-12-2014 01:03 PM

Wang, 'Low Cost Library Construction...': Low Concentration
 
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Hi All

I'm using the updated Wang et al Library construction protocol [Universal adapters and Indexed Primers + Universal primers for Enrichment]. The only major modifications I've made are adjustments for a slightly lower totRNA starting material [5ng; protocol written for 15ng]. After optimizing for adapter dimers and 400bp 'bubbles' and such, my final problem to solve is significantly low outoput.

I'm getting ~8-12 ng/ÁL, which is ≤1/5 what I was getting with the TruSeq RNA kit.

I've been submitting my samples [5-6 uniquely indexed libraries multiplexed] at 5 ng/ÁL in 40 ÁL. At the above concentrations I won't be able to make that work.

Any suggestions for increasing output or alternative submission mixtures? Do I need to worry about the issues with overamplification with single stranded template [Wang's is a strand specific protocol]? Can I just crank up the cycles [I'm doing 16 now] or will that skew the population of the library?

Thanks!

MU Core 02-13-2014 06:42 AM

The supplementary info in the article below provides a protocol for working with sub-nanomolar libraries.

http://www.nature.com/nmeth/journal/...meth.1270.html

MAdkisson 02-19-2014 02:33 PM

Thanks! I'll check that out


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