5. Later this week the concentration of your DNA extraction will be quantified u

Question

5. Later this week the concentration of your DNA extraction will be quantified using a UV spectrophotometer by measuring its absorbance in units of optical density (OD) see "Nanodrop Microvolume Quantitation of Nucleic Acids" video in Lab Activity Videos for Lab 2 on Laulima for more information). DNA absorbs light most strongly at the ultraviolet wavelength of 260 nm. The absorbance of double stranded DNA (dsDNA) at 260 nm (Az60) is used to estimate concentration, with 1.0 OD equal to a dsDNA concentration of 50 ug/ml. Using this information we can calculate the concentration of dsDNA in our extractions using the following formula: dsDNA concentration 50 ug/ml x OD260 x dilution factor Using the formula provided above, calculate the concentration of dsDNA in an extraction that was diluted 30X and had an A26o reading of 0.63 OD. Show your work. (2 points) 6. If you had eluted the DNA sample discussed in question 5 with 200 ul of elution buffer during the final step of your extraction protocol, calculate the total dsDNA yield of this extraction in micrograms (ug). Do this by multiplying your values for concentration (ug/ml) an concentration of this extraction in Question 5 and your final volume comes from the amount of elution buffer you used.) Show your work and include the correct units in your final answer. (2 points) d volume (ml) together. (Recall: You

Explanation / Answer

5) DNA yield and purity can be measured by means determining the absorbance of UV light at 260nm, i.e. , by measuring A260. 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. To ensure the numbers are useful, the A260 reading should be within the instrument's linear range (generally 0.1–1.0). Concentration of dsDNA can then calculated with the help of following formula:

dsDNA concentration = 50ug/ml x OD260 x dilution factor.

Now, as given in the question,

OD260= 0.63 and dilution factor=30X. Hence, substituting these values in the formula given above, we have,

dsDNA concentration = 50 x 0.63 x 30 = 945 ug/ml

6) Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (ml)

DNA yield (ug) = 945 ug/ml x 0.2 ml = 189 ug

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