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-   -   Long DNA isolation methods? (http://seqanswers.com/forums/showthread.php?t=68071)

hoytpr 05-05-2016 06:25 AM

Long DNA isolation methods?
 
Does anyone have a set of Minion-compatible long-read DNA fractionation methods (or links to same) that are willing to share? We have no problem getting DNA for the Minion up to 10KB, but we'd like to get up to 100KB.

Thanks!

Peter

ymc 05-05-2016 06:56 PM

http://biorxiv.org/content/early/2015/05/20/019281

This preprint claims they can generate reads over 100kb. Please let us know if you can make it work. Good luck!

thermophile 05-06-2016 08:06 AM

what are you isolating from? I've had the most success getting large DNA out of environmental samples (bacteria and fungi, so difficult to burst cells) using the old school hot SDS and CTAB lysis followed by very gentle phenol chloroform extraction. Pipet gently-maybe even using wide bore tips. I haven't tried the minion yet, I was making fosmid libraries

hoytpr 05-06-2016 09:09 AM

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

thermophile 05-06-2016 09:11 AM

Oh yeah, forgot to mention. I ran PFGE and cut out the desired size. Like I said, old school

hoytpr 05-06-2016 11:30 AM

You can't get much more "old school" than me. And proud of it!

I see this as the gold standard right now for generating ONP long-reads:

http://biorxiv.org/content/early/2015/05/20/019281 by John Urban.

If we can get this kind of distribution I'd be satisfied. But we still want to see if we can get longer reads... someday we are bound to need them.

victorace 03-16-2020 02:47 AM

Quote:

Originally Posted by hoytpr (Post 193389)
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

Hi everyone,

I'm going to start a project to sequence the genome of gram-positive actinobacteria with long-reads in a MinIon instrument.
I would like to know if you finally get a good protocol or kit for genome DNA isolation that could share, together with tips, for this purpose.

Thank you in advance

mrsrankind1990 03-27-2020 09:10 AM

Quote:

Originally Posted by victorace (Post 231664)
Hi everyone,

I'm going to start a project to sequence the genome of gram-positive actinobacteria with long-reads in a MinIon instrument.
I would like to know if you finally get a good protocol or kit for genome DNA isolation that could share, together with tips, for this purpose.

Thank you in advance

Hi. Can I contact you personally and discuss some details regarding your project?

victorace 03-30-2020 05:42 AM

Quote:

Originally Posted by mrsrankind1990 (Post 231868)
Hi. Can I contact you personally and discuss some details regarding your project?

Hi mrsrankind1990,

Yes, you can contact me personally. I'm pretty new in this forum, so I don't know how to send you a private message. I will try it by adding you to my contact list...


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