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Old 10-27-2017, 09:51 AM   #1
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.
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File Type: jpg 10.17.2017_libraries_labeled.jpg (50.0 KB, 35 views)
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File Type: pdf TapeStationTest Results.pdf (983.3 KB, 36 views)

Last edited by cpylant; 10-27-2017 at 09:56 AM.
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Old 10-27-2017, 06:12 PM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238

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.
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Old 10-30-2017, 07:48 AM   #3
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Location: Gainesville

Join Date: Oct 2017
Posts: 2

Number 2 could be the primary culprit here. I'll try submitting on a D1000 tape. Thanks for the information!
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bioanalyzer, fragment size, gbs, library preparation, tapestation

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