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Old 05-06-2016, 09:09 AM   #4
hoytpr
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Location: Stillwater

Join Date: Dec 2009
Posts: 62
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Hi,

We are starting with bacteria, gram-negative rods. I suspect getting HMW DNA won't be a problem, but getting it sized correctly will be a challenge. The Covaris g-tubes only seem to 'accidentally' produce the 100Kbp+ long reads at a very low frequency, but are the only thing on the market I can find until you go up to expensive equipment like the BluePippen.

Because we have a few (~reference) genomes that are somewhat closely related, I'm considering a restriction enzyme digest with rare-cutters to get extra long, easily repairable fragments (+1 for defined lengths!), and mixing them with the largest outputs I can get from a Covaris tube. We have an older Diagenode shearing instrument, but they just don't help people trying to develop new protocols... unless you buy a new instrument from them.

We will likely need PFGE to confirm library sizes as a Agilent Tape Station only claims accuracy to 60KB.

BUT... I was hoping someone could either share a working protocol or tell me what NOT to try. We've been with the ONP MinION access program, and while the computational people are doing great, we are hoping to improve the starting preps. Down the road we are going to be working on wheat and other crops.

Thanks,

Pete

Last edited by hoytpr; 05-06-2016 at 09:12 AM. Reason: added reference genomes
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