You\'re in a research lab and have one sample of 50.55uM of 1% MgCl2 that must b
ID: 511207 • Letter: Y
Question
You're in a research lab and have one sample of 50.55uM of 1% MgCl2 that must be tested.Your assay kit gives a range of 3-15nmol. Write out any necessary calculations to carry out this experiment. Explain why it falls within linear range.
Materials:
Magnesium Assay Buffer (25mL)
Magnesium Developer (1vl)
Magnesium Enzyme Mix (1vl)
Magnesium Standard, 150nmol/uL (0.1mL)
96 well flat-bottom plate (clear)
Spectrophotometric multiwell plate reader
Protocol:
-Dilute 10uL of Magnesium Standard with 990uL of water to prepare a 1.5nmol/uL standard solution.
Add 0,2,3,6, and 10uL of the 1.5nmol/uL standard solution into a 96 well plate, generate blank, 3,6,9,12,15 nmol/well standards. Add water to each well to bring volume to 50uL.
Sample Preparation (Remember insoluble)
-*1-50uL can be added directly to the well
-Bring samples to a final volume with water.
Assay Reaction
-Add 35 uL Magnesium Assay Buffer, 10uL Developer, 5uL of Magnesium Enzyme Mix to each of the wells
-Place wells on horizontal shaker/mix well by pipetting.
-Incubate the reaction for 10min at 37oC. Cover the plate/protect from light during this time.
Measure absorbance at 450nm at initial time (A450)initial. Must be within linear range of curve.
Incubate the plate at 37o throughout measurements. Take measurements every 5min until reach highest (A450) point at 1.5 (A450)final. Continue to protect from light.
Explanation / Answer
1. Prepare solutions having 10 fold, 100 fold and 1000 fold dilutions etc. We are doing this just to get our test results within the range. In other words we have to convert the solutions from macromolar to nanomolar concentrations.
2. Time of incubation affects the enzymatic kinetics, so the plate must be incubated in dark after regular time intervals.
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