Appendix: Using the Beckman DU-6 Spectrophotometer

June 13, 1990

Matthew S. Holt

The instructions given below are for the use of the Beckman DU-6 Spectrophotometer in quantitating and analyzing the purity of double-stranded DNA by UV spectroscopy.

Quantitating

The Beckman DU-6 Spectrophotometer can be used to get an accurate measurement of DNA concentration. DNA at a concentration of 50 ug/ml has an Absorbance260 = 1. Solving this equation for unknown concentration, the concentration (ug/ml) = A260 x 50. To conserve DNA and to get accurate readings, dilute the original sample, e.g. 1:20 in 1X TE. Hence, the final concentration equation is:
DNA concentration (ug/ml) = A260 x Dilution Factor x 50
A 1:20 dilution is ideal for samples of concentration up to 450 ng/ul (the concentration in ug/ml = A260 x 1000). If the DNA concentration appears to be higher than 450 ng/ul, dilute and mix the DNA well in TE before resampling (because the readings from too concentrated samples may not be accurate, and are difficult to handle). Minimum diluted sample volume for quantitating is 500 ul, maximum volume is 1.5 ml.
1. Turn the DU-6 Spectrophotometer on by pushing the ON/IDLE key (turn the DU-6 off by pushing this same key). Press the UV key to turn on the UV light source. The UV light source needs approximately 15 minutes to warm up and will blink at the bottom of the screen until it has completed. Once the UV message stops blinking, the UV light source is ready. If the DU-6 is left at this point for over 20 minutes, a "WARNING - LOW ENERGY" message will appear. To correct this, turn the DU-6 off by pushing the ON/IDLE once to turn off, again to restart, then press the UV key and warm up 15 minutes.
2. Prepare samples for quantitating. For a 1:20 dilution take 25 ul of DNA sample and add to 475 ul 1X TE. Samples should be vortexed hard - 15 seconds on high speed. You will also need 500 ul of 1X TE to use as a zero standard. Always use the same batch of buffer for the zero as was used to dilute the samples. If the samples are diluted in water, then use the same water for the zero.
3. Once the UV source is ready, the DU-6 will allow parameter programming for your particular samples. For multi wavelength spectrophotometry press the MULTI WAVELENGTH and the parameters will appear on the screen. Use the cursor and the keyboard to program the following parameters:

   MULTI WAVELENGTH	#01
   FUNCTION	[Abs]
   # OF WAVELENGTHS	3
   WAVELENGTH A	230.0
   WAVELENGTH B	260.0
   WAVELENGTH C	280.0
   # OF CELLS	Variable

   Be certain to push ENTER after changing any parameter. Once all parameters have been entered, press the START key, and the DU-6 will start its calibrating, which takes approximately 2 minutes.

4. After the DU-6 has finished its calibrations, the next step is to set the zero standard. Do this by pressing the AUTOZERO key. This tells the DU-6 to read the first cuvette and set that value to zero. It will only do this for the first cuvette of any run.
5. Place the zero standard in the first cuvette and the DNA samples in the other two cuvettes. Push RUN, and the DU-6 will print out on the top of the screen "RUNNING SAMPLES", and on the screen the absorbance values at each wavelength for all samples. The zero readings should be "0.000". The DU-6 will automatically move to cuvette #2 and so on for the programmed for # of cells. After it has read the third cuvette the DU-6 will print the message "INSERT SAMPLE - PUSH RUN" at the top of the screen. Remove the cuvettes, aspirate thesamples, rinse once with 1X TE, and put the next samples into the cuvettes and push "RUN". Continue for all samples.
6. When all samples have been run, push "COPY" key to get a printout of the readings. Rinse the cuvettes in ddH2O three times and then once in EtOH. Place open end down on a kimwipe to dry. Press ON/IDLE key to turn off the DU-6. During continuous quantitation, the spectrophotometer automatically prints after 14 samples have been processed to clear its screen.

Purity analysis

The DU-6 absorbance readings at various wavelengths can be used for a simple but less indicative test of DNA purity.

1. A230:A260 Ratio Peptide bonds, amino acids, and lysis buffer salts absorb light at 210 nm. This, however, is an absorbance peak because many molecules absorb at 210 nm. A230 is an absorbance valley and should be read instead. If the sample is contaminated by peptides, amino acids, or lysis buffer components, an abnormally high A210 will distort A230, giving it a slightly higher absorbance value. The calculated ratio of A230 over A260 should fall between 0.30 - 0.50 for pure DNA. A value higher than 0.50 indicates possible contamination and the DNA should be re-precipitated in EtOH and resuspended. Once resuspended, repeat the DU-6 spectrophotometry.
2. A260:A280 Ratio Phenol absorbs light at 270 nm. Its spectra has a slight shoulder which appears at approximately 272 nm. Proteins which possess tyrosine residues absorb light at 280 nm. If the sample is contaminated with any of these molecules, the absorbance at 280 nm will increase. The calculated ratio of A260 over A280 for pure DNA should fall between 1.75 - 2.10. If a DNA sample has a A260:A280 ratio outside of these limits, reprecipitate the DNA in EtOH, resuspend, and repeat spectrophotometry. (If after reprecipitating this ratio is still off, this may indicate incomplete proteinase K digestion for genomic DNA.)

References:

ABI 340A Nucleic Acids Extractor Manual