SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
DSN normalization with TruSeq kits bhennuy Sample Prep / Library Generation 23 05-11-2012 05:15 AM
Use of DSN normalization in SOLiD RNA-seq? daughart RNA Sequencing 4 03-09-2012 12:40 PM
RNA-seq library DSN normalization megalodon RNA Sequencing 2 09-01-2011 01:44 AM
DSN experts? whw Sample Prep / Library Generation 3 08-14-2011 05:29 PM
low efficiency of DSN normalization nasobema RNA Sequencing 7 05-18-2011 12:35 PM

Reply
 
Thread Tools
Old 12-02-2010, 01:15 AM   #1
henry.wood
Member
 
Location: Leeds, UK

Join Date: Apr 2010
Posts: 63
Default DSN normalization

Hello. Has anyone tried Illumina's DSN normalization protocol. We're planning to have a go using a couple of FFPE samples. Looking through the methods it seems that tight temperature control is pretty important, but I would appreciate any input from people who know any of the little tricks that never make it into the official methods.
henry.wood is offline   Reply With Quote
Old 12-03-2010, 12:26 PM   #2
mnkyboy
Member
 
Location: Seattle, WA

Join Date: Mar 2009
Posts: 87
Default

Use flip cap strip tubes this decreases the time getting them open and thus reducing the chance you lose your Cot curve.

Keep everything close and use two pipettors at a time (one in each hand), one to add the buffer or enzyme and the other to mix. This will reduce the time you have the lid off. You really want to move fast but do not want to introduce bubbles.

I think the protocol says to mix with a pipettor set at 40 ul but I do 35 ul since the 40 always introduces bubbles for me and if you remove the tube from the thermal cycler its game over.
mnkyboy is offline   Reply With Quote
Old 12-06-2010, 12:04 AM   #3
henry.wood
Member
 
Location: Leeds, UK

Join Date: Apr 2010
Posts: 63
Default

Thanks a lot, that's exactly the sort of thing I was after. It's too expensive an experiment to be messing around for ages trying to get a pilot study working due to silly beginners' mistakes.
henry.wood is offline   Reply With Quote
Old 11-19-2014, 12:36 AM   #4
blanco
Member
 
Location: Iceland

Join Date: Apr 2012
Posts: 28
Default

Hi,
all the posts here on DSN are rather old and likewise publication using the method are few and date a couple of years back.

Thus, I was wondering if people are still using DSN normalization for RNAseq libraries or is it just not worth the trouble?

Specifically I am interested in DSN treatment coupled with poly A selection in human samples in order to be better able to identify lowly expressed transcripts.

Any suggestions or comments appreciated,
thanks,
blanco
blanco 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 07:03 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO