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Old 10-17-2016, 08:33 PM   #1
JDSwenson
Junior Member
 
Location: Bay Area, CA

Join Date: Apr 2016
Posts: 8
Default Stingray mRNA Fragmentation NEBNext

Hi all,

I am using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina following mRNA isolation using dT beads.

I've been having some trouble with my mRNA fragmentation time. In this protocol, the fragmentation occurs while eluting the mRNA from the beads into a first strand synthesis buffer/random primer mix. Based on their data using universal reference human RNA, NEB suggests incubating at 94C for 15 min to generate 200 bp inserts, but I am getting 100-150 bp constantly.

Does anybody have experience using this kit and do you have any suggestions regarding how to go about optimizing the fragmentation time to generate 200 bp inserts? Next, I'm planning to fragment at 5, 7, 10, and 12 minutes to see what size my fragments are, but every time I test this it costs time and money. I'd greatly appreciate any input.

Thanks for your time,
John
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