40 | Chapter 4: Instrument Qualification
Liquid Test 3
This test verifies operation of the reader at 340 nm, and is provided for sites requiring
proof of linearity at wavelengths lower than those attainable with the Absorbance Test
Plate. This test is optional because the reader has good “front end” linearity
throughout its wavelength range.
Materials
• New 96-well, clear, flat-bottom microplate (Corning
®
Costar #3590
recommended)
Use a new microplate; any fingerprints or scratches may cause
variations in readings.
• Calibrated hand-pipette(s)
• Beakers and graduated cylinder
• Precision balance with readability to 0.01 g
• Buffer solution described below
Buffer Solution
• Deionized water
• Phosphate-Buffered Saline (PBS), pH 7.2–7.6, Sigma tablets, #P4417
(or equivalent)
• β-NADH Powder (β-Nicotinamide Adenine Dinucleotide, Reduced Form)
Sigma bulk catalog number N 8129, or preweighed 10-mg vials, Sigma
number N6785-10VL (or BioTek PN 98233). Store the powder according to
the guidelines on its packaging.
1. Prepare a PBS solution from the Sigma tablets.
2. In a beaker, mix 50 mL of the PBS solution with 10 mg of the β-NADH
powder and mix thoroughly. This is the
100% Test Solution.
3.
(Optional) Read a sample of the solution at 340 nm; it should be within
0.700 to 1.000 OD. If low, adjust up by adding more powder. Do not adjust
if slightly high.
Prepare the Plate
1. Prepare the 75% Test Solution by mixing 15 mL of the 100% Test
Solution with 5 mL of the PBS Solution.
2. Prepare the 50% Test Solution by mixing 10 mL of the 100% Test
Solution with 10 mL of the PBS Solution.
BioTek
®
Instruments, Inc.
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