SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Complete Genomics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Cell Free DNA Isolation from Urine Bioo Scientific Vendor Forum 0 05-18-2016 08:54 AM
Long DNA isolation methods? hoytpr Oxford Nanopore 5 05-06-2016 11:30 AM
DNA isolation & organelle DNA issue floem7 General 6 08-16-2013 08:50 PM
Bacterial genomic DNA isolation cheggers Sample Prep / Library Generation 0 10-04-2011 02:14 AM
Isolation of genomic DNA pDNA Sample Prep / Library Generation 0 05-10-2010 08:06 AM

Reply
 
Thread Tools
Old 01-27-2017, 01:13 AM   #1
hodap
Junior Member
 
Location: Norway

Join Date: Jan 2017
Posts: 2
Default DNA isolation methods

What approach could I take to isolate the DNA for species have high level of hybridization and polyploidy and they are from Sorbus, Rosaceae, Plant.?
in order to get highest possible yield (and no fragmentation)
and then I am going to do RAD-sequencing.
I mean is it possible to get enough and promissable output from RAD sequencing if I provided not high quality DNA extraction?
Thank you in advance
HOda

Last edited by hodap; 01-27-2017 at 03:44 AM. Reason: to explain more
hodap is offline   Reply With Quote
Old 01-27-2017, 03:42 PM   #2
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 910
Default

For RAD-Seq and related techniques average fragment length around 10Kb is sufficient. The important quality factor is lack of restriction enzyme inhibitors. You can use a commercial kit (preferably column based) or follow literature such as following:
http://www.tandfonline.com/doi/pdf/1....2015.11513205

Most (maybe all) of current RAD-Seq data analysis software have been developed for processing diploid genomes so you should be aware of the data analysis challenge with polyploid species. This will vary depending on downstream applications such as population studies or mapping.
nucacidhunter is offline   Reply With Quote
Old 01-27-2017, 05:31 PM   #3
atcghelix
Member
 
Location: CA

Join Date: Jul 2013
Posts: 74
Default

Do you often find that restriction enzyme inhibitors are a problem nucacidhunter? We sometimes have samples that drastically don't behave like others in their group and I've been wondering how often that's a problem.
atcghelix is offline   Reply With Quote
Old 01-27-2017, 05:52 PM   #4
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 910
Default

It is rare and mostly I have seen it when phenol has been used in extraction. I assume those have phenol residues.
nucacidhunter is offline   Reply With Quote
Old 01-30-2017, 01:06 AM   #5
hodap
Junior Member
 
Location: Norway

Join Date: Jan 2017
Posts: 2
Default

Quote:
Originally Posted by nucacidhunter View Post
For RAD-Seq and related techniques average fragment length around 10Kb is sufficient. The important quality factor is lack of restriction enzyme inhibitors. You can use a commercial kit (preferably column based) or follow literature such as following:
http://www.tandfonline.com/doi/pdf/1....2015.11513205

Most (maybe all) of current RAD-Seq data analysis software have been developed for processing diploid genomes so you should be aware of the data analysis challenge with polyploid species. This will vary depending on downstream applications such as population studies or mapping.
Thanks for your reply nucacidhunter I will read the literature it seems great.
hodap is offline   Reply With Quote
Old 01-31-2017, 06:28 AM   #6
ATϟGC
Member
 
Location: Canada

Join Date: Jun 2013
Posts: 30
Default

I would expect that Sorbus species would have a fair amount of polysaccharides. I have had good success precipitating plant and animal DNA by CTAB dilution. Here are two articles with protocols:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441248/

http://onlinelibrary.wiley.com/doi/1...12616/abstract

The second article used the extracts of the CTAB dilution method for PacBio,Illumina mate-pair and RADseq.

One problem I have found with these methods is that you need good amounts of DNA in the lysis buffer in order for efficient precipitation. This can be challenging if you are working with dried leaf tissue. If you get a lot of little crystals floating around after adding the dilution buffer and incubating at 60 celcisu then you are likely going to have very good yields of very clean DNA.
ATϟGC 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 02:45 AM.


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