Question

If you resuspended your DNA in 50 ul of water before you added it to the cuvette, how much DNA did your extraction yield? You should include the DNA in the 10 ul you used for the measurement when you calculate the total yield.

Answer 1

The total DNA yield, including the DNA used for the measurement, is found by calculating the original DNA concentration from the spectrophotometer reading and then multiplying by the total volume. The result is a total yield of 978 µg.

Step-by-step explanation:

To calculate the total DNA yield from your extraction, including the DNA used for measurement, you must first understand the concentration obtained from the spectrophotometric analysis. In the example provided, a dilution was made by adding 50 microliters of DNA sample to 950 microliters of water, resulting in a maximal absorbance of 0.326 at 260 nm. Utilizing the fact that the average extinction coefficient (Ɛ) for double-stranded DNA is 0.020 ng/µL-1cm-1, the concentration of the original DNA sample can be calculated.

The calculation follows these steps:

1. Apply Beer-Lambert Law: Absorbance = Ɛ * c * l, where c is the concentration and l is the path length of the cuvette (typically 1 cm). Given an absorbance (A) of 0.326, we have 0.326 = 0.020 * c * 1. Solving for c gives us c = 0.326 / 0.020 = 16.3 ng/µL for the diluted sample.
2. Calculate the concentration of the undiluted sample by factoring in the dilution, which is a 1000-fold increase (50 µL to 1000 µL). Therefore, the undiluted concentration is 16.3 ng/µL * 1000 = 16,300 ng/µL or 16.3 µg/µL.
3. Finally, to determine the total yield including the 10 µL used for the measurement, multiply the concentration by the final volume. If you added the DNA to 50 µL of water and used 10 µL for measurement (indicating a total volume of 60 µL), the total yield would be 16.3 µg/µL * 60 µL = 978 µg.