Laboratory Protocols
Quantification of DNA
Purpose
The concentration and the purity of the isolated DNA (and RNA) can be measured with a UVspectrophotometer, which requires quartz cuvettes. If this equipment is not available, there is
another method that can be used: a gel allows estimation of the concentration in comparison
with the bands of the marker lambda/HinDIII.
A: UV Spectrophotometry
Material
UV spectrophotometer
Quartz micro cuvettes or disposables appropriate for UV light (1 ml)
TE-buffer
Your isolated DNA
Method
1. Switch the UV-spectrophotometer on: follow the instructions provided for the UV-area
2. Rinse the cuvettes with TE-buffer, if you do not do this the DNA might get stuck to the
cuvette
3. Dilute 2 l of your DNA sample with 1000 l TE-buffer.
4. Fill one cuvette with this solution, use TE-buffer as a blank
5. Measure the spectrum between 230 and 320 nm, for the calculations you need to know
the extinction at 230, 260, 280 and 320 nm.
6. Rinse the cuvettes after use with water and let them leak dry on a tissue.
Calculation of the concentration DNA
a. By measuring with UV spectrophotometer
A260 – A 320 * dilution factor
20
(in this example, the dilution factor is 501)
Concentration DNA (in mg/ml) =
Purity: A260/A230 should be around 2.4
A268/280 should be between 1.8 – 2.1
Less than 1.8: there are proteins in the solution
Higher than 2.1: your solution is possibly contaminated with RNA.
Laboratory Protocols
B. Estimation of DNA concentration from ethidium bromide fluorescence
See Agarose Gel Electrophoresis further on. The DNA of the bacteriophage Lambda is 45000
bp large. HinDIII cuts this in 7 fragments, varying from 23000 to 500 base pairs. Suppose you
put 2 g lambda/hinDIII in the marker lane, then the 23000 fragment is 23000/45000 x 2 g: is
almost 1 g of the total amount. The intensity of this highest band is what you get from 1 g.
In the table you can look up how, in this example, the intensity of the band can be related to
the concentration:
Lambda/HinDIII
23000
9000
6500
4500
2300
2000
500
Fragment of total: Amount in slot: 2 g
23000/45000
9000/45000
6500/45000
4500/45000
0.2 g = 200 ng
2300/45000
2000/45000
500/45000
Compare the intensity of the band of your sample with the intensity of the bands of the
marker, and estimate the concentration. Then calculate how many g/ml you have, keep in
mind how much of your sample you loaded in the slots (normally 2 – 5 l).