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Low Qubit concentration

Qubit qPCR library prep

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#1 sw8362

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Posted 22 November 2013 - 09:14 PM

We have recently bought a Qubit from LifeTechnologies. We use dsDNA HS kit in with 195 ul working solution 5 ul DNA. Standards are prepared according to the instructions. Many of the samples we\ve measured were however lower than expected and we are worried that something is wrong.

 

1. For a few samples we had qPCR estimates from a few years back and included these from comparison. They differ considerably, though.

Sample  1 qPCR 9.1 ng/ul Qubit 2.1 ng/ug

Sample 2 qPCR 10.9 ng/ul, Qubit 3.2 ng/ul

Has someone else experienced these problems?

 

Also, we unaccidentally stored the dsDNA HS reagents in the freezer for the first 24 hours, Could that be the cause of our weird results?

 

 



#2 jerryshelly1

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Posted 24 November 2013 - 03:32 PM

What did you store your DNA in? It is possible that your samples have degraded somewhat. Try reading the concentration on a Nanodrop. If you believe your HS reagents are compromised, take a manufacturer provided control plasmid and measure its concentration on the Qubit. This should tell you if your solutions are bad.



#3 sw8362

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Posted 25 November 2013 - 11:14 AM

The extracts were stored in -20 freezers for most of the time, but many of them are fairly old (extracted c. 10 years ago), so perhaps DNA degrades with time, despite being stored in freezers. Thanks!







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