Bio-Rad Rat Diabetes Assays - Manual (Page 45)

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If mixing singleplex assays with 20x stocks of beads and detection antibodies, follow these
directions. Enter the number of wells to be used in the assay:_______
1
1. Determine the volume of 1x coupled beads needed.
a. Each well requires 50 µl coupled beads (1x): _______ x 50 µl = _______ µl
1 2
b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl
2 3
c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl
2 3 4
d. Enter the number of singleplex sets (or analytes) tubes that will be multiplexed = _______
5
e. Volume of 20x coupled beads required from each stock tube:
_______ µl / 20 = _______ µl
4 6
f. Total volume of combined bead stocks: _______ x _______ µl = _______ µl
5 6 7
g. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
4 7 8
2. Determine the volume of 1x detection antibody needed.
a. Each well requires 25 µl detection antibodies (1x): _______ x 25 µl = _______ µl
1 9
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
9 10
c. Total volume of 1x detection antibodies: _______ µl + _______ µl = _______ µl
9 10 11
d. Enter the number of singleplex sets (or analytes) that will be multiplexed = _______
5
e. Volume of 20x detection antibodies required from each stock tube:
_______ µl / 20 = _______ µl
11 12
f. Total volume of combined detection antibody stock: _____ µl x _____ = _____ µl
12 5 13
g. Volume of detection antibody diluent required: ____ µl – ____ µl = ____µl
11 13 14
3. Determine the volume of 1x streptavidin-PE needed.
a. Each well requires 50 µl streptavidin-PE (1x): _______ x 50 µl = _______ µl
1 15
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
15 16
c. Total volume of 1x streptavidin-PE: ______ µl + ______ µl = _______ µl
15 16 17
d. Volume of 100x streptavidin-PE required: _______ µl / 100 = _______ µl
17 18
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
17 18 19