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Old 02-12-2014, 01:03 PM   #1
Location: Oakland

Join Date: Mar 2011
Posts: 11
Default Wang, 'Low Cost Library Construction...': Low Concentration

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?

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