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

Question

1)    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.

A260           A280           A260/ A280            [DNA], µg/ml

Plasmid prep #1      0.244              0.135                                                                      

Plasmid prep #2      0.263              0.115   

2)    Based on the values obtained for the two plasmid preps above, which plasmid prep appears to be of higher quality and how did you come to this conclusion? What is the most likely contaminant that may result in the observed A260/A280 ratio for the lesser quality plasmid prep?

3)    After performing a plasmid prep, you find a large amount of protein/salt contaminants in your plasmid DNA. What is a possible explanation for how this could have occurred?

Explanation / Answer

1) Concentration of DNA = Absorbance value at A260 x 50ug/ml x Dilution factor

Thus, the A260/A280 ratios and concentrations for two sets of DNA preparations are as follows.

Plasmid prep#1 A260/A280 = 1.807

Concentration = 50 x 0.244 = 12.2 ug/ml

Plasmid prep#2 A260/A280 = 2.286

Concentration = 50 x 0.263 = 13.15 ug/ml. Note that there would be no dilution factor as the question requires the concentration of undiluted sample to be found.

2) Plasmid prer#1 appears to be of higher quality as the ratio is 1.8 which signifies pure DNA as per commonly available literature. Plasmid prep#2 appears to be contaminated with either RNA or free nucleotides from degraded DNA. Occasionally phenol used during isolation may also give rise to an increased ratio.

3) The solution required for high salt binding conditions may not have been mixed properly. Because of improper mixing post lysis the DNA fails to precipotate properly leading to high salt and protein contamination. Moreover the PCI method used may not be having proper fresh PCI which may not have removed contaminating proteins. The preparation may also be treated with a protease to chop off contaminating proteins.

"A malaria biologist"

Related Questions

Navigate

• Browse (All)
• Browse 1
• Subjects

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