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Determination of DNA concentration

Standard Protocols

Determination of DNA concentration using the spectrophotometer

  1. Turn on machine and wait for it to warm up
  2. Wash glass cuvettes thoroughly before use and if 2 are used then make sure they are a ‘matching’ pair.
  3. For Spec. in Dave Andersons bay then press ‘goto ?’ type in ‘260’ press ‘enter’ machine will change wavelength. Insert 1ml of water into machine and press ‘autozero’ For other machines then set wavelength to 260 and autozero with water.
  4. Put 5m l of DNA concentration? into a clean matching cuvette, add 1ml of water and mix by covering with parafilm and inverting.
  5. Insert cuvette into machine and note reading i.e. 0.030.
  6. Follow steps 3 again but this time set wavelength to 280, note reading.
  7. Repeat procedure 3 times with new samples of DNA noting readings at both A260 and A280.
  8. Take an average of the A260 readings and multiply the figure by 10 to give the concentration of DNA per ml. i.e. 0.030 x 10 = 3.0mg/ml
  9. Take average of A280 readings and divide A260 average by the A280 average this should give a figure between 1.8-2.0 if it is out of the range then the DNA is not pure and sample should be purified using phenol/chloroform.

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