quantification-qubit.md

DNA Quantification with Qubit

Theory

In order to work with DNA, we need to know how much DNA we have. Qubit uses fluorescence to measure DNA concentration. Qubit works by analyzing the fluorescence intensity of fluorescent dye binding to double-stranded DNA.

Gather Materials

• DNA Elution Plate or Tubes
• Micropipette (P10, P200)
• Pipette Tips (10uL, 200uL)
• Qubit Tubes
• Qubit Reagent Kit - dsDNA High Sensitivity

Quantification

1. Count out enough qubit tubes for the number of samples you have.
2. Label the tubes to correspond with your sample IDs.
3. Make Qubit Working Solution by adding:
   1uL x number of samples = Qubit reagent (98uL for 96 samples + 2 standards)
   199uL x number of samples = Qubit Buffer (19,502uL for 96 samples + 2 standards)
4. Add 198uL of qubit working solution to each qubit tube.
5. Add 2uL of DNA sample to its corresponding qubit tube.
6. Vortex for 5 seconds and let samples set for 1 minute on the bench.
7. Plug in qubit to turn on.
8. Select 'dsDNA.'
9. Select 'dsDNA High Sensitivity.'
10. Get 2 more qubit tubes. Add 190uL of working solution to each tube. Add:
    10uL of Standard #1 to tube 1
    10uL of Standard #2 to tube 2
11. Click 'Read Standards.'
12. Insert Standard #1 and read standard.
13. Insert Standard #2 and read standard.
14. Click 'Run Samples.'
15. Make sure the volume says 2uL and output is ng/uL.
16. Read all of your samples.
17. Record quantification in lab notebook.

Troubleshooting Your Quantification