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Old 06-15-2012, 07:32 AM   #1
Arvind Bhagwat
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Location: Beltsville,MD

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Default Optimal fragmentation of bacterial mRNA

Would any one please share conditions used for fragmenting bacterial mRNA? I am shooting for ~200 nt and starting mRNA conc. is ~75-100 ng/. I am using NEB Next mRNA library kit. They recommend 5 min at 94 C in Mg buffer. However this is for eukaryotic mRNA. I will appreciate if someone could share their success story using this kit with bacterial (E. coli/Salmonella/Bacillus ...) mRNA. I have depleted total RNA using Epicenters Ribo-Zero kit. Thanks everyone!
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Old 06-18-2012, 08:33 AM   #2
ZWB
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I've been successful using the NEBNext fragmentation kit with an incubation of 3 minutes instead of 5. Make sure to use only 200ng/rnx (or less) and pool multiple rxns during cleanup. This has worked well for total or rRNA depleted samples from a variety of Gram negative bacteria.
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bacterial rna seq, library prep, mrna fragmentation

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