Quantification of DNA using UV - Vis

From LPDwiki

Necessary Materials and Instruments

1. Biotek UV - Vis

2. 25 mM Phosphate Buffer (PB)

3. Probe/target DNA solution to be quantified

Procedure

Important:

  • Clear peak at ~ 260 nm indicates successful execution of the protocol. The yield is generally ~75% corresponding to ~ 15 - 18 μM. (The DNA concentration after reduction is ~ 75 % of that before reduction). Store at -20°C until usage.


1. Turn on Biotek UV - Vis.


2. Open Biotek program (it usually takes a while as the computer is slow).


3. Open the Take3 plate.

3a. Pipette phosphate buffer (4 µl) onto each of the micro-spots.

3b. Wipe with tek-wipe, not kim wipe as that leaves residue and causes interference with measurements.


4. For measurements, you always need a blank.

4a. Pipette 25 mM PB onto the first row (4 µl on each spot).

  • Duplicates are recommended, i.e one row for each sample/ blank (2 micro-spots).

4b. Pipette DNA samples in subsequent rows.


5. Once the Biotek program opens, choose "Nucleic Acid Quantification".


6. In the program, select ssDNA, measurement wavelength 260 nm, select microspots (blank and samples).


7. There's one more drop down menu on the top where you need to select "Take3 Plate"


8. Press eject, place the Take3 plate inside.


9. Start measurement.


10. Biotek first makes sure that blanks are fine (they turn green) and then proceeds with the measurement.


11. Results can be exported as Excel files (prompt opens up).

  • The output concentration should be in ng/μl and this should be converted to μM.