DNA quantification with PicoGreen

Prepare TE

Calculate the total volume of 1xTE needed:

• 200 µl per sample
• 100 µl per standard
• Total of 3-400 µl for the DNA standard dilution (depending on the assay you use, see step 2)
• Make enough to count for pipetting losses!!!
• E. g. for high range: 20 samples + 2* 4 standards + DNA standard the amount is 20200 + 8100 + 300 = 5100 µl -> make 5500 µl

Dilute from 20xTE stock (supplied in the PicoGreen kit).

• Volume of 20xTE = final volume/20;
• Volume of water = final volume – volume of 20*TE
• E.g. To make 5 ml: mix 250 µl of 20*TE with 4750 µl of water.

Prepare DNA standard

Dilute 100 µg/mL DNA standard (supplied in the PicoGreen kit) to working dilution. DNA standard  (supplied in the PicoGreen kit) to working dilution.

• For high range prepare 2 ng/µl: mix 6 µl of DNA stock with 294 µl of 1*TE
• For middle range prepare 0.5 ng/µl: mix 2 µl of DNA stock with 398 µl of 1*TE
• For high sensitivity prepare 0.05 ng/µl: mix first 2 µl of DNA stock with 18 µl of 1TE (working solution), then mix 2 µl of the working solution with 398 µl of 1TE

Prepare PicoGreen

Calculate the total volume of 1:200 diluted PicoGreen reagent needed.

• For each standard and each unknown sample, a volume of 100 µL will be needed = total number of samples (standard + unknowns) x100 µl + some for pipetting losses.
• e.g. For standard series of 8 and 20 samples (=28 total) prepare 28*100 µl =2800 µl: -> make 3000 µl

Dilute PicoGreen reagent with 1*TE.

• Volume of PicoGreen = final volume/200;
• Volume of TE = final volume – volume of PicoGreen
• e.g. Mix 15 µl of PicoGreen with  2985 µl of 1*TE

KEEP diluted PicoGreen IN THE DARK UNTIL USE!!!

Prepare plate - add standards

• Use  NUNC 96-well flat bottom black plates. Cover with aluminium foil inside the lid.
• Mark samples needed to be measured on the lid and also diagram your plate layout in the table as follow (When possible, measure samples at least in duplicates).
• Plan the standard curve in the microtiter plate.

Table 1 High range standard curve: for 2ul samples, test limit is 0 to 100 ng/ul - for higher concentration dilute your samples.

A	B	C	D	E	F
Plate Well	volume of TE (uL)	volume (uL) of 2ug/mL stock DNA	volume of diluted picog dye (uL)	final DNA concentration in assay (ng/mL)	total DNA in well (ng)
A1 & A2	0.00	100.00	100	1000	200
B1 & B2	90.00	10.00	100	100	20
C1 & C2	99.00	1.00	100	10	2
D1 & D2	100.00	0	100	0	0

Table 2 Medium range standard curve: for 2ul samples, test limit is 0 to 25 ng/ul

A	B	C	D	E	F
Plate Well	volume of TE (uL)	volume (uL) of 0.5ug/mL stock DNA	volume of diluted pg reagent (uL)	final DNA concentration in assay (ng/mL)	total DNA in well (ng)
A1 & A2	0.00	100.00	100	250	50
B1 & B2	90.00	10.00	100	25	5
C1 & C2	99.00	1.00	100	2.5	0.5
D1 & D2	100.00	0	100	0	0

Table 3 Low range standard curve: for 2ul samples, test limit is 0 to 2.5 ng/ul

A	B	C	D	E	F
Plate Well	volume of TE (uL)	volume (uL) of 0.05ug/mL stock DNA	volume of diluted pg reagent (uL)	final DNA concentration in assay (ng/mL)	total DNA in well (ng)
A1 & A2	0.00	100.00	100	25	5
B1 & B2	90.00	10.00	100	2.5	0.5
C1 & C2	99.00	1.00	100	0.25	0.05
D1 & D2	100.00	0	100	0	0

Prepare plate - add TE

• Add 1*TE to each standard well according to table 1-3.
• Add 95-98 µl of 1TE to each sample well. Prepare 2 wells for each sample (duplicates). I.e. if you add 2 µl of sample add 98 µl of 1TE in the case of 5 µl sample add 95 µl 1*TE (see point 6).

Prepare plate - add DNA samples

• Add 2-5 µl of sample to each sample well.

Prepare plate - add DNA standard

• Add diluted DNA standard according to Table 1-3.

Prepare plate - add PicoGreen

• Add 100 µl of diluted PicoGreen to each of the wells (both sample and standard wells) and mix by pipetting 5-10 times.

Incubate in room temperature in dark for at least 5 minutes and measure with the plate reader (ask for help using the plate reader if using it for the first time).

0h 5m 0s

To calculate your DNA concentration:

1. Create a calibration curve in excel or similar from the standards (x axis: measured fluorescens, y axis: total DNA in standard wells in ng from corresponding tables in step 4).
2. Use the equation of the fitted trendline (excel) to calculate the amount of DNA (ng) in your samples.
3. To calculate the DNA concentration of your samples (ng/μl), divide the calculated amount of DNA (ng) by the volume of DNA (μl) that you added in step 6 (2-5 μl).