Biotek Synergy HTX, Operator'S Manual

Page: 133 / 184

Absorbance Liquid Tests |
111
1.
Weigh out 0.092 g of FD&C Yellow No. 5 dye powder into a weigh boat.
2.
Rinse the contents into a 1-liter volumetric flask.
3.
Add 0.5 mL of Tween 20, or 5 mL of BioTek’s wetting agent.
4.
Fill to 1 liter with DI water; cap and shake well. The solution should
measure approximately 2.000 OD when using 200 µL in a flat-bottom
microwell.
38B
Prepare the Plate

Be sure to use a new microplate, because fingerprints or scratches
may cause variations in readings.
1.
Using freshly prepared stock solution (Solution A or B), prepare a 1:2
dilution using deionized water (one part stock, one part deionized water;
the resulting solution is a 1:2 dilution).
2.
Pipette 200 µ L of the concentrated solution (A or B) into the first column of
wells in the microplate.
3.
Pipette 200 µL of the diluted solution into the second column of wells.

After pipetting the diluted test solution into the microplate and before
reading the plate, we strongly recommend shaking the plate for four
minutes. This will allow any air bubbles in the solution to settle and the
meniscus to stabilize. Alternatively, wait 20 minutes after pipetting the
test solution before reading the plate.
39B
Read the Plate
1.
Using Gen5, read the microplate
five times
at 405 nm using the Normal
read mode, single wavelength, no blanking. Save the data after each read
(“Normal” plate position).
2.
Without delay, rotate the microplate 180 degrees so that well A1 is in the
“H12” position. Read the plate
five more times
, saving the data after each
read (“Turnaround” plate position).
3.
Print out the ten sets of raw data, or export them to an Excel spreadsheet.
Analyze the Results
1.
Calculate the mean value for each physical well location in columns 1 and 2
for the five plates read in the Normal position, and then again for the five
plates read in the Turnaround position. This will result in 32 mean values.
2.
Perform a mathematical comparison of the mean values for each microwell
in its Normal and Turnaround positions (that is, compare A1 to H12, A2 to
H11, B1 to G12,… H2 to A11). To pass this test, the differences in the
Synergy HTX Operator's Manual