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Old 09-29-2014, 10:23 AM   #1
laurynas
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Location: Oxford

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Default libraries messed up after thawing

Hi all,

I'm having some issues with my library prep. Basically I prepared the libraries and the TapeStation gel looked beautiful - no adapters, primer dimers or anything . I froze the libraries until I got access to the sequencer. Then once I thawed them, I decided to run them on the TapeStation again, to make sure they're ok - and, oh horror, it was nothing like what I saw before freezing them. I even tried heating them to 95'C for 5min and cooling to RT at 2'C/min and still - awful smear. Any idea why this is? (images attached)

I tried running those libraries on the NextSeq and apparently I have "massive" contamination with adapters and due to that there's not enough diversity in my sequence for the instrument to calibrate itself within the first runs. But there were no adapters or primer dimers on the gel visible before freezing the samples.

Very confusing any ideas/advice would be hugely appreciated!
Attached Images
File Type: png Osteo_O10-O25_libraries.png (29.7 KB, 40 views)
File Type: png Thawed.png (42.4 KB, 35 views)
File Type: png Thawed and heated.png (35.9 KB, 29 views)
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Old 09-30-2014, 06:10 AM   #2
Seqwork
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Default

Try qPCR to see what you have.
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Old 09-30-2014, 08:40 AM   #3
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Default

What library prep method did you use? How many cycles of amplification?
Also what are the spike-in standard sizes for your tapestation runs?

I can say we normally freeze libraries and don't see issues with them. Are you sure these are the same tubes you ran on the TapeStation previously?

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