SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Good Beads / Best Beads paolo.kunder SOLiD 2 03-27-2013 02:12 PM
g-TUBE for DNA Shearing coffee52 Sample Prep / Library Generation 0 02-06-2012 10:46 PM
Beads not sticking to slides pmiguel SOLiD 20 04-08-2011 11:40 AM
illumina alternative v1.5 protocol for small rna seq vs. the standard protocol ik76 Sample Prep / Library Generation 1 03-25-2010 02:24 PM

Reply
 
Thread Tools
Old 03-28-2013, 10:09 AM   #1
Gina_P
Member
 
Location: New York

Join Date: Dec 2011
Posts: 20
Default Beads sticking to tube during Ribo-Zero protocol

Hi all. I noticed that the Ribo-Zero magnetic beads tend to stick to the sides of the tube after you add your RNA sample and mix with pipetting and vortexing, and that this effect carries on through the end. When I finally pipette the 90 uL of sample from the magnetic stand, I usually just am very careful to guide the tip to the liquid without touching any sides since there will be beads stuck to it. However, recently I did the protocol and the sides of the tubes looked much cleaner than normal. My only thoughts are that I used a different brand of PCR tube during the incubation with the removal solution... any other ideas for why this may have changed? Or what your experiences are using Ribo-Zero?
__________________
- Gina
Gina_P is offline   Reply With Quote
Old 05-06-2013, 03:58 PM   #2
ribodoc
Junior Member
 
Location: orange county, ca

Join Date: Dec 2011
Posts: 6
Default

We recently used ribo-zero for metabacteria and have >50% of our reads mapping to bacillus subtilis rRNA and e. coli rRNA. It appears that the capture failed for some reason. Does anyone perform QC to check for probe leakage? Anyone else experienced probe leakage? Its pretty frustrating.
ribodoc is offline   Reply With Quote
Old 05-07-2013, 05:58 AM   #3
Gina_P
Member
 
Location: New York

Join Date: Dec 2011
Posts: 20
Default

We carried the libraries through despite the odd appearance of the beads which seems to be not unusual after all, and got less than 99.5% rRNA contamination with Oryza sativa using the plant leaf kit. I can't speak for the other kits and your application though. It would be worth it to contact Epicentre. They are very good at helping you troubleshoot until the protocol is working for you (providing some reagents for your trouble in the process).
__________________
- Gina
Gina_P is offline   Reply With Quote
Old 05-07-2013, 10:47 AM   #4
ribodoc
Junior Member
 
Location: orange county, ca

Join Date: Dec 2011
Posts: 6
Default

Thanks for the reply. We've been using the metabacteria kit for awhile with good results so our recent experience is an outlier however our level of confidence in the product has decreased. Beyond basics such as making sure the beads are used and stored under the recommended conditions, Epicentre tech support was unable to help us. We have the contaminating sequences so we'll probably use PCR to QC the ribo-zero treated RNA for the capture oligos.
ribodoc 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 11:39 PM.


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