You have purified recombinant plasmid DNA from a small culture of bacterial cell

Question

You have purified recombinant plasmid DNA from a small culture of bacterial cells. You resuspended the DNA in a volume of 50microL. You dilute 20microL of the purified DNA into a total volume of 1000microL of distilled water. You measure the absorbance of the diluted sample using spectrophotometer set at 260nm and 280n and obtained following readings:

A260=0.550

A280=0.490

a) What is the DNA concentration of the plasmid DNA in the sample you purified? (not the diluted sample)

(dilution factor is 1/50)

b) How much total DNA was purified by this procedure?

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

Please show your work including units!

Explanation / Answer

1 OD at 260 nm corresponds to 0.050 microgram/mL or 50 ng/microliter double stranded DNA.

a) Concentration of DNA at A260 = A260 * 50 * 1/dilution factor = 0.550 * 50 * 50 = 1375 ng/microliter or 1.375 microgram/mL

b) The DNA purified by this procedure = concentration of DNA * volume of the sample = 1375 ng/microliter * 50 microliter = 68750 ng or 68.750 microgram

c) A260/A280 ratio of the DNA sample = 0.550/0.490 = 1.122

For purified DNA the ratio A260/A280 should be between 1.8 and 2.0. If the the ratio is less than 1.8 or more than 2.0 corresponds to the contamination of protein or RNA respectively. In this case the sample is contaminated with the protein.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.