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-   -   Bioanalyzer/TapeStation fragment sizes larger than agarose gel (http://seqanswers.com/forums/showthread.php?t=78797)

cpylant 10-27-2017 08:51 AM

Bioanalyzer/TapeStation fragment sizes larger than agarose gel
 
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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. :confused:

nucacidhunter 10-27-2017 05:12 PM

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)

cpylant 10-30-2017 06:48 AM

Number 2 could be the primary culprit here. I'll try submitting on a D1000 tape. Thanks for the information!


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