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beetle_doc 08-21-2015 08:50 AM

Odd bioanalyzer results
 
2 Attachment(s)
I had a customer that submitted some antibody libraries which I ran on our MiSeq. The results looked good up until about 100 bases then the Q30 took a nose dive and the %A increased, suggesting that the sequence was reading into the adapter on the other side. The gel image showed a library fragment size of about 600 bp, so I had her run it on the Bioanalyzer (high sensitivity DNA). The Bioanalyzer showed a size of about 200 bp, which would be consistent with the MiSeq's poor results, but also showed a large smear at the upper marker, which doesn't show up on the gel image. Can anyone explain why the bioanayzer and agarose gel analyses would give such different results? I have attached a picture of the gel and a bioanalyzer trace of one of the samples
Thanks

pmiguel 08-21-2015 10:39 AM

The 200 vs 600 bp result, I would tend to blame on different buffer/salt concentrations in the marker( 1KB ladder -- my favorite) and the samples.

Okay, for the high molecular weight stuff visible on the bioanalyzer chip, but not your gel -- I would have to reach into deep speculation, so be forewarned... How about, that material is genomic DNA, single stranded after many cycles of denaturation during in PCR. Very poor visibility of single stranded DNA with the ethidium bromide in your gel, but a high sensitivity chip will visualize it just fine.

Irritatingly, when I originally asked whether single-stranded DNA/RNA would be visible on a high sensitivity chip, Agilient tech support answer that I should not use it for that purpose. Thanks Agilent.
Later experimentation showed that single stranded molecules were visible and migrated aberrantly with respect to their actual length. See here for more details.
--
Phillip

beetle_doc 08-25-2015 06:28 AM

Thanks for the response. I will pass the information along to my client.
Mike


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