Biology, 14.11.2019 06:31 jak000067oyyfia
•determine concentration of enzyme stock solution, if unknown, by taking an a280 nm reading of a 1: 100 dilution (in water). use a total volume of 1 ml in the cuvette.
• dilute some of the enzyme stock with buffer a to make a 4 mg/ml solution.
• serially dilute the 4 mg/ml solution with buffer a to make working solutions of 400 µg/ml and 40 µg/ml.
• prepare 30 µl of each working solution for every sample
the pi of the lab gives you a tube of enzyme and tells you the following before disappearing into the office to write more grant proposals:
➢ there is 50 µl of enzyme stock solution. the enzyme is expensive to purify, so follow the protocol exactly, using as little of the stock solution as possible.
➢ the concentration of the stock solution is currently not known, but a 1 mg/ml concentration of the pureenzyme has an a280 nm of 2.0.
➢ you’ll be performing the assay on 12 samples.
➢ make enough of each working solution so that you have at least 400 ul to work with when you do the assay (to cover any waste and/or inefficiencies in pippetting).
using the spectrophotometer to read the absorbance at 280 nm, you get a reading of 0.784.
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•determine concentration of enzyme stock solution, if unknown, by taking an a280 nm reading of a 1:...
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