SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Nextera insert sizes larger than expected pjuneja Sample Prep / Library Generation 48 06-15-2016 09:07 AM
Tapestation quality peaks show unwanted larger fragments. mzahir89 Sample Prep / Library Generation 1 08-19-2015 05:22 AM
Disagreement between bioanalyzer and agarose gel for PCR amplicon library mbirnb Sample Prep / Library Generation 4 08-03-2015 10:28 AM
rRNA sizes on Agarose gel lior lobel Sample Prep / Library Generation 1 07-19-2013 06:21 AM
Size selection: agarose gel / acrylamide gel amazonic9 Sample Prep / Library Generation 2 12-05-2012 03:56 AM

Reply
 
Thread Tools
Old 10-27-2017, 09:51 AM   #1
cpylant
Junior Member
 
Location: Gainesville

Join Date: Oct 2017
Posts: 2
Unhappy Bioanalyzer/TapeStation fragment sizes larger than agarose gel

I am preparing libraries for GBS, and although the agarose gel image is as expected (i.e. the bulk of fragments between 200 and 400bp), TapeStation result show the fragment distribution shifted 300-400bp larger. This is a 1% gel stained with EtBr (in gel, not post-stained) 100V for 35 mins (distance between electrodes = 16cm). The TapeStation results are consistently larger, and the service lab is unable to provide any explanation for the discrepancy. The core lab told us to use the TapeStation after BioAnalyzer runs showed peaks that were not present in the agarose gel (our expected fragment sizes were correct, but a second more concentrated grouping at 600-2kb was also present which left the core at a loss for explanation).

I am wondering if I should trust the gel image over the TapeStation results. Can anyone suggest why TapeStation sizes would differ so much compared to the gel image? I appreciate your time and thank you in advance.
Attached Images
File Type: jpg 10.17.2017_libraries_labeled.jpg (50.0 KB, 18 views)
Attached Files
File Type: pdf TapeStationTest Results.pdf (983.3 KB, 18 views)

Last edited by cpylant; 10-27-2017 at 09:56 AM.
cpylant is offline   Reply With Quote
Old 10-27-2017, 06:12 PM   #2
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 1,083
Default

I would trust the size indicated by gel. Three issues with Tape:

1- Tape has expired and sometimes that will affect the sizing accuracy
2- The used Tape (HS D5000) accuracy for sizing short fragments is very low. Those should be run on HS D1000 tape.
3- Markers on some is misidentified (not the main issue in this case)

Last edited by nucacidhunter; 10-27-2017 at 06:17 PM.
nucacidhunter is offline   Reply With Quote
Old 10-30-2017, 07:48 AM   #3
cpylant
Junior Member
 
Location: Gainesville

Join Date: Oct 2017
Posts: 2
Default

Number 2 could be the primary culprit here. I'll try submitting on a D1000 tape. Thanks for the information!
cpylant is offline   Reply With Quote
Reply

Tags
bioanalyzer, fragment size, gbs, library preparation, tapestation

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 02:24 PM.


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