SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
MiSeq Wash question Krina796 Illumina/Solexa 10 02-24-2016 09:29 AM
FLX Maintenance Wash Aniki 454 Pyrosequencing 23 10-25-2013 12:36 AM
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
454 Maintenace wash bia 454 Pyrosequencing 1 01-21-2009 01:22 PM

Reply
 
Thread Tools
Old 10-13-2016, 11:39 AM   #1
salyamoff
Junior Member
 
Location: Russia

Join Date: Sep 2013
Posts: 4
Default SureSelect Wash 2

Hi everyone! I had a failure with sureselect exome enrichment - no DNA in captured sample. I reread manual, then i read Faircloth (2013) "Target Enrichment of Illumina Libraries" and Holmberg (2005) "The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures" and i see that:
1) Samples need be washed 30 minutes total at 65C. (Manual)
2) Wash 2 buffer is 15 mM NaCl (Faircloth)
3) On this conditions streptavidin-biotin interaction will be broken with 100% chances. (Holmberg)
I don't think that SureSelect protocol is unreliable, but i think that my problem due to washing step.

Last edited by salyamoff; 10-13-2016 at 09:15 PM.
salyamoff is offline   Reply With Quote
Old 10-15-2016, 07:12 AM   #2
seqdavis
Junior Member
 
Location: Midwest

Join Date: Sep 2015
Posts: 5
Default

Hello salyamoff,

We currently use Agilent's SureSelect XT and QXT protocols on a regular basis and have come across this a few times. Here are a few questions for your troubleshooting:

1. What was your target yield (ng) going into hybridization? We have found and the literature will tell you that one needs at least 250ng of pre-capture library going into hybridization. If this is not the case, you will have to adjust your bait ratio.

2. Did you perform the initial 95C denaturing step prior to the addition of your baits? If this doesn't occur, there are no single stranded molecules for the bait to anneal to therefore, no material will get 'pulled down.'

3. During your washing at 65C, did you make sure the beads were resuspended prior to the 10 minute incubation (which is done 3 separate times)?

4. Did you use water anywhere in the washing?

Take a look at these sections of your experiment and see if anything stands out. From my experience, this is where the majority of issues arise with the SureSelect protocols.
__________________
SeqDavis
seqdavis is offline   Reply With Quote
Old 10-20-2016, 09:16 AM   #3
salyamoff
Junior Member
 
Location: Russia

Join Date: Sep 2013
Posts: 4
Default

I repeated a enrichment and capture with another beads. Sequensing was done good. It looks like my dynabeads was depraved. But this beads succesfully captured biotenylated proteins recently. I think it because protein capture conditions aren't so extreme.
salyamoff is offline   Reply With Quote
Reply

Tags
exome enrichment, hybridization, hybridzation wash, sureselect

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 01:42 AM.


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