Quantifying DNA w/ Spectrofluorometer

The spectrofluorometer is a useful device for measuring DNA concentration. It is better to use the spectrofluorometer than to measure 260 nm absorption on the spectrometer as that technique will quantify all nucleic acids (DNA + RNA). The fluorometer used a double stranded binding dye which detects DNA preferentially.

Safety Note: The dye (bisbenzimide/Hoechst No.33258) is a mutagen. Please wear gloves when quantifying DNA.

This technique is useful for quantifying DNA to an accuracy of about 20%. Essentially you can find out if you have a lot or a little. Because samples are compared to a calf standard, consistency is the key to getting good results.

1. Warm up machine for about 15 minutes by turning power on. Occasionally you will get an error message when the fluorometer goes through it's initial electronic checks. Open the cuvette cover and close it again. This usually fixes the problem.

2. Make up enough working dye solution so you have 4 ml for each sample.

To make 200 ml, mix 20 ml of 10X TNE from refridgerator
180 ml of water
20 ul of Hoechst stock solution (1 mg/ml) also on door of fridge
This solution will keep for a few weeks. A bottle of made up working dye is also kept in the fridge.

3. Use calf thymus DNA standard which is 100 ng / ul. This standard will keep for many months. It's nice to use the same standard over time as a given tube of standard is remarkably reproduceable but there is tube to tube variation.

4. Fill glass cuvette with 2 ml of working dye solution from the pump bottle. Wipe outside of cuvette clean with kimwipe. The cuvette is marked on the side (with a G or a 4H). Place the cuvette in the spectrofluorometer to read a zero. It is helpful to face the marking in the same direction every time. This compensates for any inconsistency in the glass. Push the zero button.

5. Add 2 ul of the calf thymus standard to the sample. Set up a consistent routine such as mixing with the pipette five times and then using parafilm to mix the entire contents of the cuvette five times. (Wipe outside of cuvette again if necessary. Put the sample back in the machine and take a reading (push Calibrate). You are then prompted for the concentration of the standard which is typically 100 (ng/ul).

6. Pour out the cuvette and rinse once with working dye solution. Refill with new solution. Recheck calibration and repeat as ncessary till reproducible. There may be some zero drift, especially at first, but usually the machine settles down.

7. Now you are ready to measure your samples. Add 2 ml of solution to the cuvette. Insert it and push zero. Add 2 ul of your sample. Push read. This will give you the sample reading in ng / ul.

8. Repeat ad infinitum.

9. For cycle sequencing of plasmids, it works best to use 500 ng. Based on the measured concentration, determine the volume of sample to use. Alternatively, dilute all samples to the same concentration and then use the same volume in cycle sequencing. This has the disadvantage that if you dilute them all to say 50 ng/ul and then find they are too dilute, it is difficult to concentrate them again.