PicoGreen Protocol

Items Needed

• PicoGreen assay kit (ThermoFisher P7589)
• Clear-bottom black plate (Griener 675096)
• Molecular-grade water
• Aluminum foil
• Access to plate reader (Striepen Lab)

Procedure

1. Prepare DNA
1. Thaw, vortex and spin down your DNA plate(s).
2. (optional) Create a dilution plate for high-concentration samples. Stool is diluted 1:10 - 1:20.
2. Prepare PicoGreen Reagent
1. (if needed) Make TE from 20x stock: 2.5 ml 20x TE + 47.5 ml water.
2. Dilute PicoGreen reagent 1:200 in a foil-wrapped 50 ml tube.

Note: use within a few hours of preparation

# Plates	1x TE	PicoGreen Reagent
1	6 ml	30 ul
2	12 ml	60 ul

3. Prepare Standard
1. Create 2 ug/mL lambda DNA standard (if needed, good for 1 month at -20C):
1. 490 ul TE
2. 10 ul 100 ug/mL lambda DNA
2. Make standard curve as follows, if needed (good for 1 week at -20C):

*Final concentration after diluted PicoGreen reagent added at 1:1 ratio.

Std	Well	2 ug/ml Std (uL)	TE (uL)	[Final] (ng/mL)*
1	A1	250	250	500
2	B1	100	400	200
3	C1	50	450	100
4	D1	25	475	50
5	E1	10	990	10
6	F1	5	995	5
7	G1	1	999	1
8	H1	0	1000	0

4. Prepare Plates
1. Add 50 ul diluted PicoGreen reagent (from step 2) to each well for every sample and standard.
2. Standards:
1. Add 50 ul of each standard to wells in column 1 as indicated in step 3b.
3. Samples:
1. Add 48 ul TE to all sample wells.
2. Add 2 ul sample DNA (or DNA dilution) to all sample wells.
5. Cover plate with foil.
6. Vortex on slow speed for 10 seconds.
7. Incubate 2-5 min protected by light.
8. Read on plate reader at 480 nm excitation.
9. Export results to Excel document.
10. Analyze using the Excel analysis template.