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-   -   Weird bioanalyzer results (http://seqanswers.com/forums/showthread.php?t=41614)

exo 03-12-2014 11:34 PM

Weird bioanalyzer results
 
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Hey all,

Yesterday, for the first time ever i was about to slam my head into the bioanalyzer. I ran 8 samples prepared with nexteraxt protocol after pcr cleanup and size selection using 29ÁL of ampure xp beads. The results did not make ANY sense at all. The presence of HUGE fragments that are even bypassing the upper ladder! The concentration of the samples on qubit was the following:
Sample 1 = 2.05
Sample 2 = 2.55
Sample 3 = 3.96
Sample 4 = 7
Sample 5 = 3
Sample 6 = 4.75
Sample 7 = 6.68
Sample 8 = 7.3

if those HUGE fragments are truly present, than the concentration doesnt really correlate with them does it?
Has anyone seen this phenomena using the hs DNA chip?

Best,
Exo

Simone78 03-13-2014 09:03 AM

Hi!
It might simply be that you have some contaminants that are driving the Bioanalyzer crazy. We see this kind of weird things all the time and in our case is due to traces of ethanol left over from the bead purification or to an excess of salt in the elution buffer (HS chips tolerate a max of 10 mM Tris as buffer, according to the specs). Also some leftover detergent from the previous steps can interfere and screw up the chip.
Since all the samples are run on the same lane of the Bioanalyzer, when one goes bad the others also tend to go bad, especially the one that is immediately after the bad one.
Good luck with your troubleshooting!

DRYTCYV 03-13-2014 02:50 PM

Hi,

In a Illumina webminar they showed something like this one, https://www.dropbox.com/s/eom2x2u78i...2022.45.44.png

You can try to clean-up again, but if its an artifact will stay there.

mcampo86 03-27-2014 02:15 PM

The smaller the ratio of ampure beads used, the larger the fragment you will obtain.


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