In: Chemistry
You are provided with the following portion of a protocol:
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 pure enzyme 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 pipetting).
3. (6 pts) Figure out your plan for making each solution required in the protocol. Be certain you FINISH with the required volumes of EACH solution! a. Calculate what is needed to make the 4 mg/ml solution b. Calculate what is needed to make the 400 ug/ml working solution c. Calculate what is needed to make the 40 ug/ml working solution
a) You haven’t provided the concentration of the stock solution. The PI handed you 50 µL of the enzyme stock and you prepared a 4 mg/mL enzyme solution from the same. However, your enzyme stock must have been more concentrated (concentration > 4 mg/mL; otherwise you cannot dilute the concentrated solution to prepare the diluted solution.). I need to know the concentration of the stock.
b) I will assume that you have a 4 mg/mL enzyme solution. Suppose you prepare 50 µL of 400 µg/mL enzyme solution by dilution of the 4 mg/mL enzyme solution. Use the dilution equation.
M1*V1 = M2*V2 where M1 = 4 mg/mL; M2 = 400 µg/mL and V2 = 50 µL.
Plug in values:
(4 mg/mL)*V1 = (400 µg/mL)*(1 mg/1000 µg)*(50 µL)
===> (4 mg/mL)*V1 = (0.4 mg/mL)*(50 µL)
===> V1 = (0.4*50)/(4) µL = 20 µL.
Take 20 µL of the 4 mg/mL solution and add (50 – 20) µL = 30 µL DI water to prepare the 400 µg/mL standard solution.
c) We have 50 µL of 400 µg/mL standard solution and we wish to prepare 400 µL of 40 µg/mL standard solution. Use the dilution equation again.
M1*V1 = M2*V2 where M1 = 400 µg/mL; M2 = 40 µg/mL and V2 = 400 µL.
Plug in values:
(400 µg/mL)*V1 = (40 µg/mL)*(400 µL)
===> V1 = (40*400)/(400) µL = 40 µL.
Take 40 µL of the 400 µg/mL solution and add (400 – 40) µL = 360 µL DI water to prepare the 40 µg/mL standard solution.
Note that the overall dilution is (4 mg/mL)/(40 µg/mL) = (4 mg/mL)*(1000 µg/1 mg)/(40µg/mL) = 100; i.e, the stock 4 mg/mL solution has been diluted 100 times and hence the desired 1:100 dilution is achieved.