Biotek Synergy H1, Operator'S Manual

Page: 151 / 190

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Stir the solution (preferably using a stir table) until the PBS is completely
dissolved.
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Check the pH; it should be between 7.2 and 7.6 at 25°C.
2. Prepare the sodium fluorescein stock solution:
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Add 2.0 mL of the buffer solution to the 1 mg Sodium Fluorescein (SF) vial. This
yields a 1.3288 mM stock solution.
l
Ensure that the dye has completely dissolved and is well mixed.
3. Carefully prepare the dilutions. Label each with “SF” and the concentration:
Mix this SF solution: with buffer: to make:
0.53 mL of 1.3288 mM stock solution 13.47 mL 50.2 µM
110 µL of 50.2 µM SF 13.89 mL 400 nM
3.5 mL of 400 nM SF 10.50 mL 100 nM
0.46 mL of 100 nM SF 13.54 mL 3.3 nM Corners Test
4.24 mL of 3.3 nM SF 9.76 mL 1 nM Sensitivity/Linearity Tests
Fluorescence Polarization (FP) Test:
As described in the Materials section, the recommended test solutions are available
from Invitrogen Corporation (or BioTek). They do not require additional preparation.
Time-Resolved Fluorescence (TRF) Test:
As described in the Materials section, the recommended test solutions are available
from Invitrogen Corporation (or BioTek).
l
Shake the FluoSpheres container vigorously for 30 seconds prior to pipetting.
Alternatively, sonicate or vortex the container.
l
Mix 10 μL of FluoSpheres with 10 mL of deionized water, in a 15-mL conical-bottom,
polypropylene sample tube. This yields a 20-nM equivalent suspension.
l
Shake the vial vigorously for 30 seconds prior to pipetting. Alternatively, sonicate or
vortex the container.
l
Mix 10 μL of 20-nM suspension with 10 mL of deionized water, in a 15-mL conical-
bottom, polypropylene sample tube. This yields a 20-pM equivalent suspension.
l
Refrigerate any unused portions of the FluoSpheres. The temperature must be
b2°C to +6°C.
The prepared TRF plate can be kept for a maximum of seven days, if
covered and stored in the dark b2°C to +6°C. Allow the plate to sit
at room temperature for approximately 15 minutes prior to use. Shake the
plate gently prior to the read.
Synergy H1 Operator's Manual
Fluorescence Liquid Tests | 125