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-   -   bioanalyzer and qubit disagreement (http://seqanswers.com/forums/showthread.php?t=92841)

smsestes 02-05-2020 01:46 PM

bioanalyzer and qubit disagreement
 
We prepare clinical samples with a formal SOP and do not regularly see irregularities. Today we have a capture pool of samples that quanted at 24.0ng/uL on qubit. To determine fragment size we also run sample pool in duplicate on bioanalyzer DNA1000. Duplicate 1, there is noise around 40bp (<1ng/uL) and no peak in the expected range (300-400). Duplicate 2, there is noise around 40bp and a wide peak between 700 and 1500 (upper marker). We reran the sample pool (4 replicates) on the bioanalyzer with only the noise around 40bp.

Qubit concentration = normal
bioanalyzer fragment size = missing?

Any ideas what is going on? Where is the missing peak?

JakobHedegaard 02-06-2020 03:24 AM

Perhaps you didn't load enough on the Bioanalyzer to get a signal from a smear using a D1000 chip? Try to run on HS DNA chip.

Noveline 02-06-2020 04:50 AM

DNA 1000 Kit is for 5-50 ng/ul of DNA
Hs DNA Kit: "NGS sheared DNA or libraries: For accurate determination of DNA concentration, the total DNA in the samples must be between 100pg/μL to 10 ng/μL."

I think that in HS dna chip will be overloaded...

Check if everything is ok with gel dye mix in DNA 1000 Kit.

smsestes 02-06-2020 06:03 AM

Quote:

Originally Posted by Noveline (Post 230930)
DNA 1000 Kit is for 5-50 ng/ul of DNA
Hs DNA Kit: "NGS sheared DNA or libraries: For accurate determination of DNA concentration, the total DNA in the samples must be between 100pg/μL to 10 ng/μL."

I think that in HS dna chip will be overloaded...

Check if everything is ok with gel dye mix in DNA 1000 Kit.

Hi Noveline,

When we reran the sample pool, we included a previous sample pool which ran normally and as expected. Thus it is unlikely to be the BioA or its reagents.


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