PicoGreen dsDNA Assay for Proliferation
DEPC-treated, sterile, distilled Water (Fisher BP561-1)
Sterile PBS (Cellgro through Fisher 21-040-CV)
Nunc Nalgene 96-well black plates
Quant-iT PicoGreen dsDNA Kit (LifeTech Invitrogen P7589) Reagents stable at 4o for at
least 6 months; for longer storage, place at -20o.
1. Dilute TE Buffer with DEPC-tx, sterile, distilled water; this is supplied as a 20x solution.
The volume needed depends on the number of wells.
2. Aspirate culture medium from wells.
3. Rinse 3x in sterile PBS, removing as much as possible after last rinse.
4. Add 1000L 1x TE Buffer to each well (24-well format).
5. Label plate timepoint.
6. Freeze at -80o overnight at minimum.
1. Make a platemap (96-well, duplicate samples & stds).
2. Thaw sample plates at room temperature. Allow PicoGreen reagent to warm to room
temp (protected from light) before opening vial. (It’s likely at 4o.)
3. Label empty 1.7mL DNase-free tubes “A”-“E” for standards.
4. Dilute 20x TE buffer using sterile, distilled, DNase-free water.
(# of wells x 100L + 3.5mL for all standards)
5. Keep in mind that the working solution:
a. Must be used within a few hours of preparation
b. Must be protected from light
c. Must be kept in plastic, not glass
6. Prepare working solution:
a. Wrap conical tube in foil to protect from light.
b. Stock is 200x in DMSO. Dilute in 1xTE. Keep at room temperature temporarily.
(# of wells x 100L)
7. Prepare DNA standards:
a. Stock is 50x (100g/mL) prior to making standards for curve. Dilute 12L of
50xstock in 588L 1xTE. This is now standard “E” (2g/mL).
b. Dilute remainder of DNA standards with TE as follows:
(High-Range Standard Curve)
1xTE
“E” DNA Std
1xPicoGrnSoln Final Conc
E 0
500
500
1g/mL = 1000ng/mL
D 450
50
500
100ng/mL
C 495
5
500
10ng/mL
B 999
1
1000
1ng/mL
A 500
0
500
blank
8. Load 100L of each standard in duplicate onto a black 96-well plate. Triturate
experimental samples. Load 100L of each sample in duplicate onto plate according to
platemap.
9. Add 100L of the PicoGreen working solution to each well using a multipipettor.
10. Incubate at room temperature, protected from light, for 2-5 minutes.
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11. Measure sample fluorescence using a fluorescence microplate reader with excitation
wavelength of ~480nm and emission wavelength of ~520nm. Set max sensitivity to the
wells containing the highest standards (1000ng/mL).
12. Subtract the blank from all wells. Generate the standard curve. Find the concentration of
each sample. Graph using Excel.
13. Discard the plate. Place kit components at 4o or -20o for long-term storage.
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