You have purified 50 ul of recombinant plasmid DNA from a sm

You have purified 50 ul of recombinant plasmid DNA from a small culture of bacterial cells. You measure the absorbance of the sample using a spectrophotometer set at 260nm and 280 nm and obtain the following readings: A260=0.466 and A280=0.389

a. What is the DNA concentration of the plasmid DNA in your purified sample. Show your work throughout (including units). Hint. The conversation factor for double-stranded DNA is 1.0 OD260= 50 ug dsDNA/ ml

b. How much total DNA was purified by this procedure? Show your work.

c. What is the A260/A280 ratio of the DNA sample and what does this tell you about your preparation?

Solution

In this experiment Dilution factor(amount of DNA sample used to determine the absorbance) is not mentioned. We assumed that absorbance was determined using 2ml of cuvette. For that we have assumed that 10 ul of DNA sample from total(50ul) sample used to determine.

10ul(DNA sample)+1990 ul(TAE Buffer for dilution)

10ul/2000ul=0.005

Dilution factor = 1/0.005 = 200

(A260)DNA concentration = A260 X 50ug/ml X 200

= 0.466 X 50ug/ml X 200

= 4.466 mg/ml

(A280)DNA concentration = A280 X 50ug/ml X 200

= 0.389 X 50ug/ml X 200

= 3.89 mg/ml

A280 used to find the amount of protein contamination.

A260/A280 used to find the Purity of DNA which should be around 1.8

from this experiment A260/A280 = 1.20(may be beacuse of contamination)