1. Add 200 ul of PBS to tubes with 3x flies and homogenize.
2. Serial Dilute 50 ul of standard.
50 ul = 2 ug/ul = Std07
25 ul of Std07 + 25 ul PBS = 1 ug/ul = Std06
25 ul of Std06 + 25 ul PBS = 0.5 ug/ul = Std05
25 ul of Std05 + 25 ul PBS = 0.25 ug/ul = Std04
25 ul of Std04 + 25 ul PBS = 0.125 ug/ul = Std03
25 ul of Std03 + 25 ul PBS = 0.0625 ug/ul = Std02
25 ul of Std02 + 25 ul PBS = 0.03125 ug/ul = Std01
3. Put 10 ul of Blanks (BL = PBS); Standards (Std01-07); and Sample (Sa01-40) into 96-well plate as illustrated below. Each blank/standard/sample has a technical replicate:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | BL | Std01 | Std02 | Std03 | Std04 | Std05 | Std06 | Std07 | Sa01 | Sa02 | Sa03 | Sa04 |
B | BL | Std01 | Std02 | Std03 | Std04 | Std05 | Std06 | Std07 | Sa01 | Sa02 | Sa03 | Sa04 |
C | Sa05 | Sa06 | Sa07 | Sa08 | Sa09 | Sa10 | Sa11 | Sa12 | Sa13 | Sa14 | Sa15 | Sa16 |
D | Sa05 | Sa06 | Sa07 | Sa08 | Sa09 | Sa10 | Sa11 | Sa12 | Sa13 | Sa14 | Sa15 | Sa16 |
E | Sa17 | Sa18 | Sa19 | Sa20 | Sa21 | Sa22 | Sa23 | Sa24 | Sa25 | Sa26 | Sa27 | Sa28 |
F | Sa17 | Sa18 | Sa19 | Sa20 | Sa21 | Sa22 | Sa23 | Sa24 | Sa25 | Sa26 | Sa27 | Sa28 |
G | Sa29 | Sa30 | Sa31 | Sa32 | Sa33 | Sa34 | Sa35 | Sa36 | Sa37 | Sa38 | Sa39 | Sa40 |
H | Sa29 | Sa30 | Sa31 | Sa32 | Sa33 | Sa34 | Sa35 | Sa36 | Sa37 | Sa38 | Sa39 | Sa40 |
4. Add 50 ul of Activator to every 5 ml of Reagent. Invert gently 3-4 times.
Let sit for 15 min at room temperature before use.
After 15 min, add 190 ul Activator/Reagent solution to each well of plate.
Let plate sit at least 10 min (30 min okay) at room temperature.
5. Turn on SpectraMax M2 machine and computer.
6. Press "drawer" button to open/close plate drawer and load your plate.
7. Open SoftMax Pro software. If "no port selected" choose Com1.
Click 'plate' to read plate, 'cuvette' to read cuvette
Save as
Settings
aborbance
wavelength: 500 nm
endpoint
automix
OK
Template
unknown
concentration ug/ml
standards
1st blank
group
new group setting
name = sample
Results
Result Interpx(Plot#1@Graph#1,Values)
R2 should be ~1
y = absorbance, x = concentration, B = slope
y = A+Bx => x = (y-A)/B
TGtotal/corrected fly weight = ug/mg = plot this value