Determination of DNA concentration by Spectrophotometric Estimation

 

DNA easily dissolves in aqueous solutions. However, at high concentrations (10 mg/ml and above), dissolved DNA is viscous. At lower concentrations, one cannot detect the DNA by sight or by noting the viscosity of the solution.

When working with DNA, it is often important to know its concentration (recall that concentration will be expressed in units of mass per volume). The easiest means of determining DNA concentration is through spectrophotometric analysis. Since nitrogenous bases absorb UV light, the more concentrated the DNA solution, the more UV light it will absorb.

Rule: The concentration of pure double-stranded DNA with an A260 of 1.0 is 50 mg/ml. Thus, one can use the following formula to determine the DNA concentration of a solution.

Unknown (mg/ml)/ Measured A260 = 50 (mg/ml)/ 1.0 A260

Since there is a linear relationship between absorbance and DNA concentration, we can use some simple algebra and reformulate as follows:

Unknown mg/ml = 50 mg/ml x Measured A260 x dilution factor (see below)

 

Concentration determination (the instructor will help in the use of the spectrophotometer).

1. Obtain two falcon tubes and label one 'B' (for blank) and the other 'D' (for DNA).

2. Add 2 ml of TE buffer to the B' tube and 1.99 ml of TE buffer to the 'D' tube. [In order for the spectrophotomter to work, light must pass through the liquid. However, given the design of the machine and the cuvettes, light will not be able to pass through the liquid if the volume is less that 2 ml.]

3. Add 10 ml of your DNA solution to the 'D' tube. Mix by gentle pipeting.

4. Transfer the entire contents of the B tube to a glass cuvette in the proper slot of the spectrophotometer (which is set at A260). Since there is no DNA in this tube, the reading should be close to 0. If not, set the spectrophotometer so it reads 0. You have now calibrated the machine.

5. Using a plastic pipet, remove the water from the B tube and immediately replace it with the contents of 'D' tube. Wait several seconds and allow the A260 reading to stop drifting. When the reading becomes stable, write down the A260. This is the value you will use in the above formula.

The dilution factor

Note that you have actually measured a solution of DNA which is more dilute that your original sample. This is done mostly to conserve DNA. Isolating and purifying DNA takes time, and since the cuvettes are not sterile, you don't want to waste 2 ml of your DNA on a concentration determination! So, you use small volumes (5-20 ml), and given the constraints of the equipment, this means you have no choice but to dilute the DNA. Also, absorbance values above 2.0 are not very reliable, thus measurements in this range are likely to be very rough estimates.

In our experiment, we took 10 ml of the DNA solution and diluted it to a final volume of 2 ml (2000 ml). Thus, we diluted it by a factor of 0.005. When you measured the DNA concentration, you obtained an absorbance value of the diluted solution, but you want to know the concentration of your original solution (the one you would work from when doing experiments). To do this, you must multiply your determined concentration by the reciprocal of this dilution factor (1/0.005). Or, to put it simply, just use the following formula:

Unknown mg/ml = A260 x 50 mg/ml x 200

After doing this, hand in the following information on a separate piece of paper:

Number of tube used =

A260 =

mg/ml =

Now you know how to determine the concentration of DNA!