McClean:Qubit

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Contents

Overview

Small amounts of DNA can be measured with high sensitivity and specificity using the Qubit Fluorometer. A small amount of sample (1 - 20 μL) is mixed with a dye which fluoresces when bound to double stranded DNA (dsDNA). The Qubit quantifies the DNA by measuring the amount of fluorescence in the sample. The advantage to this approach over using the Nanodrop is that contaminates such as RNA or protein may affect the Nanodrop reading, but will not affect the Qubit reading, even with low amounts of DNA.

We have two assay kits for measuring dsDNA:

  • Qubit dsDNA BR Assay kit for quanitification of genomic or miniprep DNA
  • Qubit dsDNA HS Assay kit for quantification of PCR product or viral DNA

Materials

The dsDNA Assay kit contains the following reagents:

  • dsDNA BR reagent (3005A, small chemicals box)
  • dsDNA BR buffer (3005A, small chemicals box
  • dsDNA BR Standard #1 (0 ng/μL) (3005, 4°C fridge, small vials box)
  • dsDNA BR Standard #2 (100 ng/μL) (3005, 4°C fridge, small vials box)

In addition, you will also need

  • 1 - 20 μL DNA sample (100 pg/μL - 1 μg/mL)
  • 500 μL thin wall tube (3005, in Qubit drawer)
  • Eppendorf or other disposable plastic container

Procedure

This protocol assumes you are using the dsDNA BR Assay kit.

Sample Preparation

  1. Label the caps of the 500 μL tubes. You will need 1 tube for each sample + 1 tube for each of the 2 standards
  2. Dilute the 200X dsDNA BR reagent into the dsDNA BR buffer in the Eppendorf (or other plastic container)
    • You will need ~200 μL per reaction (1 per sample + 1 for each of the 2 standards)
    • For example: If you have 3 samples you will prepare 5 total reactions, so add 5 μL dsDNA BR reagent to 995 μL buffer
  3. Aliquot the diluted reagent into your reaction tubes. The final volume (after adding DNA) should be 200 μL
    • You should add 190 μL to each of the standard tubes
    • You are aiming to add 2 - 1,000 ng DNA in 1 - 20 μL to the reaction
    • Avoid adding small volumes of DNA (e.g., <5 μL). If your sample is concentrated, dilute and add a larger volume
  4. Add 10 μL of the standards to the appropriate reaction tubes
  5. Add the appropriate volume of sample DNA to the sample tubes
  6. Vortex briefly to mix
  7. Microfuge briefly to remove bubbles
  8. Incubate at room temperature for at least 2 min.

Measurement

  1. Plug in the Qubit Fluorometer 2.0 to turn it on
    • The other Qubit also works, but has a different menu system
  2. Select DNA
  3. Select dsDNA Broad Range
  4. Press Yes
    • The Qubit automatically saves the last standards. It is recommended to re-calibrate every time you dilute the reagent because small differences in reagent concentration may affect the results
  5. Insert the Standard #1 reaction tube, close the lid and press Read
  6. Insert the Standard #2 reaction tube, close the lid and press Read
  7. Insert a sample tube, close the lid and press Read
  8. The Qubit shows the concentration of the diluted sample (not the stock sample). To calculate the concentration of the stock, press Calculate Stock Conc.
  9. Select the volume of sample that you added to the reaction tube
  10. Change the units by pressing on the displayed units
  11. Press Save to save the stock concentration in the results
  12. When ready, insert a new sample tube and press Read Next Sample
  13. After you have finished measuring your samples, you can save your data to a USB drive or read it directly from the machine

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  • The reaction is temperature sensitive! Make sure all reagents are at room temperature and avoid warming the reaction tubes with your hands before measurement.
  • Leaving the tube in the Qubit may raise the temperature and lead to unreliable results. If you need to re-measure a sample, remove it from the Qubit for 30 s before measuring it again.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

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or instead, discuss this protocol.


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