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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/µL, 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/µL in 40 µL. 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!
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/µL, 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/µL in 40 µL. 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!
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