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Old 08-22-2013, 04:23 PM   #1
vramani
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Location: Bay Area

Join Date: Mar 2013
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Default Maximum input material for Nextera library prep?

Hi all,

I've seen some threads from people struggling with the size distribution of Nextera-generated libraries, and noticed that some people mention changing the amount of enzyme/amount of input DNA to skew the size distribution to favor longer fragments. I was wondering what experience, if any, users may have had trying 2-5 ug (or even more!) input DNA with the Nextera kit (perhaps while testing size distributions).

I've hit a point in my experiments where my current input (50 - 100 ng) results in undersampling, and would like to max out the reaction conditions to include as much input gDNA as possible...If anyone has done this, did you find that more enzyme was necessary to properly generate the library? I'm less concerned with size distribution (gel slices have worked fine for me so far), and am more concerned with overloading the tagmentase.

Thanks!
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