Quantification of DNA

1. Prepare a sample of diluted DNA. The final volume should be 400 µL but the dilution ratio will vary from 1:100 to 1:400.

2. Turn on the spectrophotometer (and the UV bulb) for at least 15 minutes to allow it to warm up. When the machine is ready, set the wavelength for 260 nm.

3. Blank the spectrophotometer by using 400 µL of the same solvent used to dilute your DNA. You will need to fill both of the quartz cuvettes (these are very expensive so please be careful with them) with this solvent.

4. Empty the cuvette and put your diluted DNA sample in the cuvette.

5. Record the OD260.

6. The concentration of your original dsDNA stock solution can be determined using this formula:

OD260 x 50 µg/ mL x dilution ratio (e.g. 100 or 400) = concentration of DNA

7. Clean the cuvettes with distilled water and turn off the spec.

Note: For many years people thought you could judge the quality of DNA by measuring a 260:280 ratio. This has been shown to be incorrect if the DNA has been cleaned with phenol, since phenol also absorbs at 260 nm. We do not do this any more.

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