Question

You purified the plasmid from an overnight cell culture and dissolved the plasmid DNA in 100 μL 0.1M Tris buffer (pH8). To quantify the plasmid, you took an absorbance measurement at 260 nm of a 1:50 dilution sample. The absorbance was 0.35. What is the concentration of the undiluted plasmid in micrograms per mL?

Answer 1

Ans. The average extinction coefficient of a double stranded DNA is 0.020 ug mL^-1cm^-1 that gives the value of 50.0 ug/ mL DNA concentration for absorbance of 1.000 at 260 nm.

So, an absorbance of 1.000 at 260 nm indicates 50 ug/mL DNA- it is taken as the standard or reference.

Concentration of DNA solution with absorbance of 0.350 at 260 nm=

(Abs. of test solution / Abs. of standard) x concentration of standard.   

            = (0.350 / 1.000) x 50.0 ug/ mL

            = 17.5 ug/ mL

Thus, the concentration of 1:50 diluted sample = 17.5 ug/ mL

Now,

Concentration of original undiluted solution = concentration of diluted sample / dilution factor

                                    = (17.5 ug/ mL) x (1 : 50) = (17.5 ug/ mL) x (1 / 50)

                                    = 875 ug/ mL

Thus, concentration of undiluted plasmid = 875 ug/ mL