Measuring DNA&RNA concentration using UV-Vis spectroscopy

Measuring DNA&RNA concentration using UV-Vis spectroscopy

Materials Needed

1. DNA sample
2. TE buffer or nuclease-free water (for dilution if necessary)
3. UV-Vis spectrophotometer
4. Quartz cuvettes (typically 1 cm path length)
5. Pipettes and tips
6. Gloves and lab coat (for safety and contamination prevention)

Protocol

1. **Preparation of the DNA Sample:**
   - If your DNA sample is concentrated, dilute it with TE buffer or nuclease-free water to a suitable concentration. Typically, a dilution factor of 1:50 or 1:100 is used.
   - Mix the sample thoroughly by pipetting up and down or gently vortexing.

2. **Blank Preparation:**
   - Fill a quartz cuvette with TE buffer or nuclease-free water. This will be used to blank the spectrophotometer.
   - Ensure there are no bubbles in the cuvette and that it is clean on the outside.

3. **Spectrophotometer Setup:**
   - Turn on the UV-Vis spectrophotometer and allow it to warm up (typically around 15 minutes).
   - Set the wavelength to 260 nm for DNA measurement. Also, set the wavelengths to 280 nm and 230 nm for purity assessment.

4. **Blanking the Spectrophotometer:**
   - Place the blank cuvette into the spectrophotometer.
   - Close the lid and press the "blank" button to zero the instrument at 260 nm.
   - Repeat this process for 280 nm and 230 nm if your instrument requires separate blanking for each wavelength.

5. **Measuring the DNA Sample:**
   - Remove the blank cuvette and carefully pipette the DNA sample into a clean quartz cuvette. Ensure there are no bubbles.
   - Wipe the outside of the cuvette with a lint-free tissue to remove any fingerprints or residues.
   - Place the cuvette into the spectrophotometer.
   - Measure the absorbance at 260 nm, 280 nm, and 230 nm.

6. **Recording and Calculating Concentration:**
   - Record the absorbance values (A260, A280, and A230).