You prepared 2 plasmids by a mini-prep method, diluted the final solutions 1:50,

Question

You prepared 2 plasmids by a mini-prep method, diluted the final solutions 1:50, and using a spectrophotometer and standard cuvette, you determined the UV absorbances at 260 nm and 280 nm shown in the table below. Calculate the ratios of A260/ A280 for your diluted samples and the apparent DNA concentrations for the undiluted plasmids, based on the formula in the manual. (20 points)

   A260    A280    A260/ A280            [DNA], µg/ml
Plasmid prep #1   0.244   0.135          
Plasmid prep #2   0.263   0.115              

Explanation / Answer

A 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. Abnormal 260/280 ratios usually indicate that a sample is contaminated by residual phenol, guanidine, or other reagent used in the extraction protocol, in which case the ratio is normally low. Inaccurate ratios may also be encountered at very low concentrations (< 10 ng/µl) of nucleic acids.

A260 A280        A260/ A280        [DNA Purity], conc.µg/ml
Plasmid prep #1   0.244   0.135       1.80                   Pure DNA       610µg/ml
Plasmid prep #2   0.263   0.115          2.286                                      657.5µg/ml

Absorbance readings are performed at 260nm (A260) where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution.

DNA concentration is estimated by measuring the absorbance at 260nm, multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA.

DNA Concentration (µg/ml) = A260 reading × dilution factor × 50µg/ml

Note:(diluted the final solutions 1:50,here the dilution factor is 50

for Plasmid prep #1 the DNA Concentration (µg/ml)=0.244× 50× 50

for Plasmid prep #1 the DNA Concentration (µg/ml)=610µg/ml

for Plasmid prep #1 the DNA Concentration (µg/ml)=0.263× 50× 50

for Plasmid prep #1 the DNA Concentration (µg/ml)=657.5µg/ml

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