SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
SureSelect QXT: Transposons and 90-minute hyb ECO Sample Prep / Library Generation 1 08-02-2015 12:25 PM
96-Well Manual Hyb Wash using Agilent SureSelect XT vspotlo1 Sample Prep / Library Generation 2 09-15-2013 12:34 PM
Agilent SureSelect XT Capture vs. SureSelect XT2 Capture ? What's the difference ? medalofhonour Sample Prep / Library Generation 4 08-07-2013 09:40 AM
Do you freeze hyb cocktails? jlove Illumina/Solexa 8 09-13-2012 05:13 PM
ChIP and SDS jdub Epigenetics 3 11-04-2010 01:30 PM

Reply
 
Thread Tools
Old 03-11-2016, 09:09 AM   #1
Squall
Member
 
Location: Ireland

Join Date: Feb 2015
Posts: 15
Default SureSelect Hyb#4 - SDS

Hi all,

Could anyone tell me the role of SDS (Hyb#4 buffer I'm assuming) in the hybridisation reaction of SureSelectXT?

It is causing us some problems and we were wondering how disastrous it would be to titrate it back, or do away with it altogether.

Thanks!
Squall is offline   Reply With Quote
Old 03-11-2016, 01:57 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,226
Default

SDS destabilises bonds between probes and library fragments (one of hybridization stringency factors) thereby reduces non-specific hybridization. Omitting it from hyb reaction would result in lower on target capture.
nucacidhunter is offline   Reply With Quote
Old 03-13-2016, 09:39 AM   #3
Squall
Member
 
Location: Ireland

Join Date: Feb 2015
Posts: 15
Default

Thanks nucacidhunter.

Do you think that it is a linear response? So if we reduced the SDS by 20, 40, 80% etc, that the reduction in on-target would increase accordingly? Or is the SDS present in excess in the reaction?
Squall is offline   Reply With Quote
Old 03-13-2016, 02:51 PM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,226
Default

I have not seen any formula to calculate the effects. I do not think that it is linear. One way to determine the effect empirically would be to reduce %SDS to the highest that downstream application can tolerate. If off-target levels are high then one can increase hybridisation temperature by 2C to compensate for reduced SDS content. Google searching DNA hybridization kinetics or dynamics should retrieve some related papers.
nucacidhunter is offline   Reply With Quote
Old 03-15-2016, 05:32 AM   #5
melinn21
Junior Member
 
Location: Boston, ma

Join Date: Jan 2015
Posts: 1
Default

I am also experiencing issues with this. While our data seems to be coming out ok, I'm finding that if I heat and then cool the hyb buffer mix (as the protocol says to do if precipitation forms) I somehow lose volume and never have enough to add to the probes (even with 5% overage). It's very strange. If I measure the volume while its still warm I have the correct volume if I measure while RT it is not enough and the precipitate has essentially returned.
melinn21 is offline   Reply With Quote
Old 04-05-2016, 06:59 AM   #6
Squall
Member
 
Location: Ireland

Join Date: Feb 2015
Posts: 15
Default

Thanks hunter for suggestions, will cycle them into the testing plans!
Squall 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 04:51 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO