Qubit Quantification Protocol

version 1.0: 2023-06-14

Objective

This is a quick step-by-step protocol for using the Qubit fluorometer with either the BR (broad range) or HS (high sensitivity) dsDNA kits. Qubit provides a much more accurate reading of DNA concentration than the Nanodrop spectrophotometer and is an essential tool for library preparation.

Reagent/Assay	Assay range	Sample starting concentration range
Qubit dsDNA HS Assay	0.2–100 ng	10 pg/μl–100 ng/μl
Qubit dsDNA BR Assay	2–1,000 ng	100 pg/μl–1 μg/μl

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Protocol

1. Pull out Qubit standards from fridge and allow to warm to RT (~5 min). Mix by gently inverting each container several times.

2. Pull out Qubit buffer and reagent (both stored at RT). Gently mix buffer by inverting several times. Reagent should be stored in dark box and exposed to light as little as possible.

Starting with v.4 of the Qubit instrument, new versions of the dsDNA kits have been released. Originally, both HS and BR kits required user to mix buffer with the respective dye to prepare the Qubit master mix. For the new 1X dsDNA HS and 1X dsDNA BR kits, a bottle of Qubit master mix is provided containing the buffer already mixed with dye. We still recommend user to make aliquots from original bottle to avoid contamination issues.

3. Calculate how much buffer and reagent you will need with the following calculations:
   • Buffer: (#samples + 2 standards + 1 for error) x 199 μL.
   • Reagent: (#samples + 2 standards + 1 for error) x 1 μL.

For Qubit v4 instrument, a Reagent Calculator button is present on the home screen, allowing user to add # of samples, # of standards, and select box for 1 extra essay to account for pipetting error.

4. Combine buffer and reagent into an appropriate-sized tube and vortex for 10 sec. This is your Qubit mix.

5. Pull out enough Qubit tubes for your samples plus 2 standards. Leave the lids open. Make sure to use the correct Qubit tubes. These need to be optically clear, don't autoclave them!

6. Pipet 190 μl of Qubit mix into the 2 standard tubes. It is not necessary to change tips between tubes.

7. Pipet 10 μL of standard 1 into one of the standard tubes and pipet 10μL of standard 2 into the other. Close and label the standard tubes “S1” and “S2” or any other code. Suggestion: use a simple dot or double dot for those.

8. Pipet 198 μl of Qubit mix into each of the sample tubes. It is not necessary to change tips between tubes.

You can run an essay with as little as 1uL of sample and up to 20uL. We recommend using 2uL if you have some sample to spare as pipetting 1uL can be cumbersome. On the other end, avoid using up too much of your sample.

8. Using a separate filter tip, pipet 2 μL of sample into the sample tubes. Make sure to insert the pipet tip into the Qubit mix to insure that all 2μL is dispensed. Close and label all of the sample tubes.

9. Vortex all tubes for 5 solid seconds.

10. Incubate all tubes for 3 minutes in the dark (drawer or cabinet).

11. Turn on the Qubit device. Select the appropriate kit. Click “yes” that you would like to use new standards. We recommend recalibrating instrument anytime you have the chance. Insert standard 1, close the lid, and press “read”. Insert standard 2, close the lid, and press “read”.

12. Insert your first sample and press “read”. After reading, press “calculate stock concentration” and adjust the sample volume to how much sample you have added in μL and the units to ng/μL. Record the quantity and click “save”.

13. Repeat step 12 for all of your samples.

You can retrived all the readings from instrument with a flashdrive. However, we recommend log readings right away to a working spreadsheet or lab notebook.

Supplies

• Qubit Fluorometer, Life Technologies
• Qubit dsDNA HS or BR Assay Kit (or cheaper alternatives like Biotium)
• 0.5ml PCR Tube, Natural