Question

3) You have four tubes containing the same DNA at the same concentration in each tube (pH 7.5) but under different environmental conditions (listed below). The TM for this DNA molecule is 78°C at physiological salt conditions (~100 mM NaCl): a. DNA in an aqueous solution that has been digested completely to nucleotides with DNAse I enzyme. b. DNA in an aqueous solution containing no salt and has been heated to 78°C. c. DNA in an aqueous solution containing physiological concentrations of salt and heated to 78°C. d. DNA in an aqueous solution containing physiological concentrations of salt and is heated only to 37oC. Match the samples above with their corresponding OD260 readings. Next, explain your reasoning for each of the matches you made. (For example, “The DNA in sample (a) gives the highest OD reading because…”). • OD260 = 0.620 • OD260 = 0.960 • OD260 = 0.475 • OD260 = 0.271

Answer 1

a. DNA in an aqueous solution that has been digested completely to nucleotides with DNAse I enzyme. P. D. 260 = 0.960

THIS sample has highest OD because this sample has free nucleotides as it digested by DNAse enzyme. And free nucleotide absorbed maximum 260 wavelength.

b. DNA in an aqueous solution containing no salt and has been heated to 78°C. O.D. 260 = 0.620

DNA is heated without salt , there will be sepration of both the stands and they will absorb increse 260 wavelength but less than free nucleotide. Salt cover the phosphate sugar backbone of DNA and decrease electrostatic repulsion in between chain. But in absence of sakt , both the strand will easily get seprated as we increse the temperature of solution.

c. DNA in an aqueous solution containing physiological concentrations of salt and heated to 78°C. O.D. 260= 0.475

as this tube contain salf so here DNA is more stable than above two tubes , but still we are heating tube at 78 degree. This will lead some of denaturation of DNA thus increased absorbance.

d. DNA in an aqueous solution containing physiological concentrations of salt and is heated only to 37oC O.D. 260 = 0.271

Here DNA present as it present in natural conditions, normal salt and temperature. So it will absorb least 260 wavelength compare to above tubes. Here DNA is present in intact form and there is no denaturation of DNA.

Finally this is the whole conclusion

Absorption of 260 wavelength order is

Nucleotides > denaturated DNA > intact DNA with out salt > intact DNA with salt.

More free surface to absorb 260 wavelength, more will be the O. D.