DNA quantification

1.I want to know whether any other reasons are there for not getting the exact OD values.
2.i also like to know why the OD value changes and lot of variation was observed if the same sample was read for second time in an spectrophotometer.

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DNA Quantification by Spectrophotometer (use for RNA free samples)


Principle for DNA Quantification:
If a DNA sample is free of contamination from protein, phenol, agarose, or RNA, its concentration can be measured accurately by determination of the amount of UV radiation that is absorbed by the bases present in an aliquot of the sample.

Equipment:
UV spectrophotometer—Ultrospectrum 2000 (Amersham pharmacia biotech)

Procedure for DNA quantification by Spectrophotometer:

1.Make sure the spectrophotometer’s UV light source has been turned on and allow it to warm up for 10 minutes before using.

2.Pipet 50 ml of dH2O into cuvette (path length: 10mm) to be used.

3.Set spectrophotometer for DNA measurement, then wavelength to be measured for 260 nm and 280 nm. Follow instructions in the instrument manual. Insert cuvette.

4.Zero spectrophotometer using cuvette containing dH2O as a blank, then set reference.

5.Pipet 2 ml of sample into an 0.5 ml Eppendorf tube containing 48 ml dH2O. Mix DNA by vortexing.

6.Remove dH2O from blank cuvette with a Pasteur pipet.

7.Pipet sample into cuvette and record OD260 and OD280 of sample.

8.Wash cuvette by rinsing with dH2O several times or rinsing with cuvette washing device. Replace cuvette in holder.Repeat process to record all samples' OD.

Calculations

For calculation of DNA concentration of samples free of RNA, the following conversion factor is used: 1 OD260 = 50 mg of DNA/ml.

DNA concentration in mg/ml can be calculated as follows:

OD260 x 50 mg DNA/ml x Dilution Factor
mg DNA/ml = ___________________________
1000 ml/ml

With a dilution factor of 25 (i.e., 2 ml in 48 ml), this formula reduces to :

mg DNA/ml = OD260 x 1.25

Results:
OD260/OD280 =1.7 -1.8

A value out of this range is not acceptable. It may indicate that the DNA sample is not in solution or that there are contaminants (i.e., protein) in the sample that may inhibit subsequent reactions. The DNA should be purified or re-extracted using phenol/chloroform/isoamyl(25:24:1) again.

Place sample in DNA container box and store in a 4°C refrigerator.

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