Need clarifications on the preparation, purification and concentration measurement of calf thymus DNA solution?

23 November 2022 1 9K Report

My objective is to prepare pure CT-DNA solution. For that, I have taken a known amount of CT-DNA fibers (sodium salt) in a 10 mM Tris buffer solution and stirred at low rpm at 4 degrees overnight. The next step, I have to purify by dialysis and here I dialyzed the solution for 24 h against the same buffer.

I'm hoping the steps so far has been correct (as the A260/280 ratio came to be 1.87). Now I need to measure the concentration of the dialyzed CT-DNA solution.

If you guys could provide a step-by-step protocol I can carry out to determine the concentration, It would be very useful.

Thank you!

Ahmad Al Khraisat

There are different methods to measure the concentration of DNA, but one common and easy way is to use a spectrophotometer to measure the absorbance of the solution at 260 nm and calculate the concentration based on the Beer-Lambert law. Here's a step-by-step protocol you can follow:

Prepare a blank solution by adding the same buffer you used for dialysis to a cuvette and set the spectrophotometer to zero absorbance at 260 nm.

Pipette a small volume (e.g. 1 µL) of the CT-DNA solution into another cuvette and measure the absorbance at 260 nm. Record the value.

Using the A260 of the sample, calculate the concentration of the CT-DNA solution in ng/µL using the following formula:DNA concentration (ng/µL) = A260 x dilution factor x extinction coefficientThe dilution factor is the ratio of the final volume to the volume of the sample added to the cuvette. The extinction coefficient is a constant that depends on the DNA sequence and is usually around 50 ng/µL cm at 260 nm.For example, if you diluted the sample 10 times before measuring, the dilution factor is 10, and the A260 is 1.0, the DNA concentration would be:DNA concentration = 1.0 x 10 x 50 = 500 ng/µL

Repeat the measurement and calculation a few times to ensure consistency and accuracy.

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