DNA Quantification using Qubit Fluorometer
The Qubit Fluorometer is a device from Thermo Fisher Scientific used to quantify DNA, RNA, and protein in samples by using a fluorescent dye that binds to these molecules. The Qubit uses specialized assay kits, like the Qubit dsDNA HS (High Sensitivity) Assay Kit, for quantifying double-stranded DNA (dsDNA).
Here is a step-by-step process of using the Qubit fluorometer for DNA quantification:
Materials Required: Qubit fluorometer, Qubit dsDNA HS assay kit (or similar), Qubit assay tubes, Pipettes and tips, and DNA samples.
1. Prepare Qubit working solution: The working solution is made by diluting the Qubit dsDNA HS Reagent in the Qubit dsDNA HS Buffer. The standard dilution is 1 part reagent to 199 parts buffer. Mix the reagent before use as it may be prone to forming precipitates.
2. Prepare DNA Standards: The Qubit assay kit includes two DNA standards (Standard #1 and #2). These standards are used to calibrate the fluorometer. Add 190 µL of the working solution to each standard tube and then add 10 µL of the respective standard. Mix gently, then read the standards on the Qubit fluorometer.
3. Prepare DNA samples: Add 198 µL of the working solution into an assay tube for each DNA sample. Then, add 2 µL of the DNA sample. Mix the solution gently.
4. Measure DNA Standards and Samples: Place the tube containing Standard #1 into the Qubit and close the lid. On the screen of the Qubit, select “DNA” then “High Sensitivity” (or the appropriate assay) and tap “Read”. The fluorometer will read the standard and then prompt you to insert Standard #2. Then, proceed to read your samples.
5. Interpret the results: Once your sample is read, the Qubit will display the DNA concentration in your sample on the screen. The Qubit fluorometer software also allows you to export your data for further analysis.
Remember to follow good lab practices to prevent contamination of your samples. In addition, to maintain the stability of the fluorescent dye reagent, avoid extended periods of light exposure.