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Qubit quantification too low yield DNA - (Aug/20/2013 )

Hi everyone!

We bought a Qubit to quantify DNA and RNA.

Today I did the first measurement and realized that the yield of DNA was too extremely low.

sample1: 1,97 ng/mL

sample2: 0,75 ng/mL

The same measure in nanodrop was 75000ng/mL and 67000ng/mL (using the same units, since nanodrop give the values by ng/uL; so 75ng/uL and 67ng/uL), respectively.

Is that correct?

I know the nanodrop overestimates the quantity, but like this?

I did not use the recomended tube. I used thin walled 0,5 mL tube (as recomended) but from Eppendorf. It means something?

Can anyone share some information about it?

Thank you in advance.

-thinker-

Which assay did you use? I.e. the broad range or the high-sensitivity?

Anyway two ideas:

I'd use only the recommended tubes bec with standard Eppendorf tubes you have no idea about light transmission, turbidity etc (i.e. perhaps most of the fluorescence signal was absorbed by the tubes leading to such low values)...or ask someone from the company if it's okay to use standard tubes.

Errors with dilution when you prepared the DNA standards for calibration?

-hobglobin-

Which assay did you use? I.e. the broad range or the high-sensitivity?

Anyway two ideas:

I'd use only the recommended tubes bec with standard Eppendorf tubes you have no idea about light transmission, turbidity etc (i.e. perhaps most of the fluorescence signal was absorbed by the tubes leading to such low values)...or ask someone from the company if it's okay to use standard tubes.

Errors with dilution when you prepared the DNA standards for calibration?

I used the high-sensitivity assay.

I considered the dilution mistake so I repeated twice. Same results.

Interestingly, I checked the raw measurements of the standards. The measure of standard #2 (100 ng/uL) is about ~10000, so I think the tube it is not the problem.

Anyway, I just buy the proper tube and I will wait it to repeat the measurement. But I am still worried with the low yield. It is too low.

-thinker-

A third idea is that you measure with nanodrop everything that absorbs light at 260 nm (DNA, RNA, even small fragments such as oligos), with Qubit you measure only DNA with the specific dye (no idea if the qubit dye distinguishes between small and large DNA fragments).

So you might have really a very low DNA concentration which is highly "contaminated" with RNA.

-hobglobin-

A third idea is that you measure with nanodrop everything that absorbs light at 260 nm (DNA, RNA, even small fragments such as oligos), with Qubit you measure only DNA with the specific dye (no idea if the qubit dye distinguishes between small and large DNA fragments).

So you might have really a very low DNA concentration which is highly "contaminated" with RNA.

About that:

I had already used the sample 1 to do a qPCR. For this purpose, I diluted the sample #1,  75ng/uL (measured previously in nanodrop) to 5ng/uL and used 4 uL (total of 20ng) of this solution to make a qCPR and worked fine. The ct was high, but worked.

If the Qubit is correct, so in fact I used 1,97ng/mL diluted 16 times (75 to 5 ng/uL), so I finaly used 0,123125ng/mL which means that I used 0,000123125ng/uL, so 0,0004925ng of DNA. If I am correct I would actually used 492,5 femtogram of DNA. I do not know if is possible to do a PCR with this amount of DNA.

-thinker-