SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Oxford Nanopore



Similar Threads
Thread Thread Starter Forum Replies Last Post
DNA isolation & organelle DNA issue floem7 General 6 08-16-2013 09:50 PM
Bacterial genomic DNA isolation cheggers Sample Prep / Library Generation 0 10-04-2011 03:14 AM
DNA isolation & library quality control Nak Sample Prep / Library Generation 2 05-25-2011 01:39 PM
Isolation of genomic DNA pDNA Sample Prep / Library Generation 0 05-10-2010 09:06 AM

Reply
 
Thread Tools
Old 05-05-2016, 07:25 AM   #1
hoytpr
Member
 
Location: Stillwater

Join Date: Dec 2009
Posts: 35
Default 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
hoytpr is offline   Reply With Quote
Old 05-05-2016, 07:56 PM   #2
ymc
Senior Member
 
Location: Hong Kong

Join Date: Mar 2010
Posts: 495
Default

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!
ymc is offline   Reply With Quote
Old 05-06-2016, 09:06 AM   #3
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 201
Default

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
thermophile is offline   Reply With Quote
Old 05-06-2016, 10:09 AM   #4
hoytpr
Member
 
Location: Stillwater

Join Date: Dec 2009
Posts: 35
Default

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 10:12 AM. Reason: added reference genomes
hoytpr is offline   Reply With Quote
Old 05-06-2016, 10:11 AM   #5
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 201
Default

Oh yeah, forgot to mention. I ran PFGE and cut out the desired size. Like I said, old school
thermophile is offline   Reply With Quote
Old 05-06-2016, 12:30 PM   #6
hoytpr
Member
 
Location: Stillwater

Join Date: Dec 2009
Posts: 35
Default

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.
hoytpr is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 11:44 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO