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Qubit Standard 2 Green RFU value variation

Qubit DNA Assay Troubleshooting

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#1 My5pam

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Posted 04 September 2013 - 06:20 AM

I am using the Qubit high-sensitivity kit to quantify low concentration samples of DNA. I am using a new standard curve with each assay, and the Qubit saves the RFU values from these points each time.

 

I have noticed that the Std 1 RFU values are relatively stable (usually around 39) on one machine, but fluctuate a bit more on another machine located in another lab (41 to 57). Std 2 RFU values are not stable on either machine (from 6000-11,000 or 6000-33,000) on each machine, respectively).

 

The ambient conditions in the labs for each machine are relatively constant and temperature is not fluctuating more than +/-3°C in each.

 

Does anyone have an explanation for this huge variation?



#2 jerryshelly1

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Posted 12 September 2013 - 09:45 AM

Are you waiting long enough in between each reading (mutiple readings on same sample)? It seems like you understand the issue with temperature and concentration reading.  I would try using the standard with each machine and then taking a manufacturer provided plasmid and reading the concentration on each machine.  This could tell you which machine is right, if both are wrong or if an undergrad left the standardizing solutions at 25C for a week.

 

Probably not the case, but open the Fluorometer lid and check and see if you see any dried solution on the spec's lenses. Dried liquid will disrupt the emitted wavelength and give you funky results.

 

If you have an additoinal spec laying around (nanodrop), verify the standard concentrations. Each standard will have a predetermined concentration in them (i.e., standard; 0-200ng/uL). This may confirm if someone has "double dipped" into your standards.

 

Good Luck


Edited by jerryshelly1, 12 September 2013 - 09:46 AM.






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