TITLE: DNA quantification with PicoGreen
AUTHORS: Anna Székely

[DESCRIPTION]
We use the Quant-iT™ PicoGreen® dsDNA kit for quantification of both extracted and amplified DNA.
The Quant-iT™ PicoGreen® dsDNA reagent is a proprietary, asymmetrical cyanine dye.  Free dye does not fluoresce, but upon binding to dsDNA it exhibits a >1000-fold fluorescence enhancement.  PicoGreen is 10,000-fold more sensitive than UV absorbance methods, and highly selective for dsDNA over ssDNA and RNA.
For moreinformation see: https://www.thermofisher.com/order/catalog/product/P7589

[BEFORE_START]
Take out PicoGreen (supplied in the PicoGreen kit) from fridge to thaw, before starting.
Protect it from light.
 
PicoGreen is solid at 4°C and thaws at room temperature. The reagent is light sensitive and should be kept wrapped in foil both while thawing and in the diluted stage.

[GUIDELINES]
IMPORTANT:
 
When quantifying non-amplified DNA (for example DNA extracted from filters), work in the prePCR area of the lab using the kit designated for prePCR.
When measuring amplified DNA (for example PCR products), work in the postPCR usinf the kit located there. 
 
QUALITY ASSURANCE:
 
Standards: always measure your samples with appropiate standards. The concentration of all of your sample has to fall within the standard range.
Replicates: always measure both for your samples and for the standards at least 2 technical replicates.
Kit properties: Always write down the LOT number of the kit you use.

[STEPS]
1. Prepare TE
 
 Calculate the total volume of 1xTE needed: 
200 µl per each technical replicate of your samples
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 20*200 + 8*100 + 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.
2. Prepare DNA standard
 
 Dilute 100 µg/mL 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 0.2 µl* of DNA stock with 399.8 µl of 1*TE 

*As pipetting of 0.2 µl is not accurate with our pipettes, it is recommended to prepare first a 10-times dilution of the DNA standard and use 2 µl of that into 398 µl of 1*TE for the high sensitivity DNA standard dilution.
3. 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!!!
4. 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.
 
 
 
Table 2 Low range standard curve: for 2ul samples, test limit is 0 to 25 ng/ul
 

 
Table 3 Low range standard curve: for 2ul samples, test limit is 0 to 2.5 ng/ul
 
5. Prepare plate - add TE
 
Add 1*TE to each standard well according to table 1-3.
Add 98 µl of 1*TE to each sample well. Prepare 2 wells for each sample (duplicates).
6. Prepare plate - add DNA samples
 
Add 2-5 µl of sample to each sample well. 
7. Prepare plate - add DNA standard
 
Add diluted DNA standard according to Table 1-3. 
8. 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.
9. 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).
5 min
10. To calculate your DNA concentration:
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).
Use the equation of the fitted trendline (excel) to calculate the amount of DNA (ng) in your samples.
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).