TITLE: Qant-iT™ PicoGreen® dsDNA Quantification
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following protocol is intended for the quantification of double-stranded DNA using </span><a href="https://www.thermofisher.com/order/catalog/product/P7589?ICID=search-p7589#/P7589?ICID=search-p7589" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Quant-iT™PicoGreen</span><span style = ":;vertical-align:super;">®</span><span style = ":;"> dsDNA Assay Kit (ThermoFisher</span></a><span>). This protocol is a simplified and condensed version of the </span><a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fmp07581.pdf&title=UXVhbnQtaVQgUGljb0dyZWVuIGRzRE5BIFJlYWdlbnQgYW5kIEtpdHM=" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">full protocol</span></a><span> from the manufacturer. The procedure described here is for 96 reactions. If samples are run in duplicates, then this should allow quantifying 40 samples.</span></div></div>

[STEPS]
?. [Prepare reaction]
Take out all reagents from the fridge and bring them to room temperature.Take out the DNA samples from the freezer. DNA samples should be slowly thawed on ice.
Quant-iT™PicoGreen® dsDNA reagent is dissolved in dimethylsulfoxide (DMSO), which freezes below 19 °C. The reagent must be completely thawed before using it by bringing it to room temperature. After the reagent thawed, it is advisable to briefly vortex the tube to make sure it is adequately mixed and to spin it down in a centrifuge or a mini centrifuge.
Quant-iT™PicoGreen® dsDNA reagent is light sensitive and should be protected from light at all times.
?. [Prepare reaction]
Prepare 22 ml 1X TE buffer by pipetting 1.1 ml of 20X TE buffer into 20.9 ml of nuclease-free water into a sterile and nuclease-free 50 ml tube.Mix by inverting the tube several times.
[20X TE buffer]
[nuclease-free water]
?. [Prepare reaction]
For high-range quantification:Dilute the DNA-standard stock solution (λ DNA 100 ng µl-1) to a final concentration of 2 ng µl-1 by mixing 10 µl λ DNA-standard stock solution with 490 µl 1X TE buffer.For low-range quantification:Prepare a 40-fold dilution of the 2 ng µl-1 DNA-standard work solution by mixing 5 μl of the 2 ng μl-1 DNA-standard work solution with 195 μl 1X TE buffer to yield a 0.05 ng µl-1 DNA-standard work solution.
[λ DNA-standard stock solution]
[1X TE buffer]
[diluted DNA-standard solution]
[1X TE buffer]
?. [Prepare reaction]
If needed, prepare a dilution of each sample in 1X TE buffer so that the reading will be within the dynamic range.
It is advisable to run samples in duplicates for a more accurate quantification
?. [Prepare reaction]
Prepare PicoGreen® work solution: 9950 µL 1X TE buffer + 50 µL PicoGreen® into a sterile and nucleic-acids free 50 ml tube. Mix and protect from light.
[1X TE buffer]
[PicoGreen®]
?. [Prepare reaction]
Prepare the following standard mixture in the first two columns of the black, sterile, 96-well plate:  ABCD1Assay versionDiluted DNA std. (µl)1X TE buffer (µl)Final DNA amount (ng)2High-range (1-200 ng µl-1)10002003Use 2 ng μl-1 standard50501004
1090205
19916
010007Low-range (50 pg µl-1 - 5 ng µl-1)100058Use 0.05 ng μl-1 standard50502.59
10900.510
1990.0511
01000
ABCD1Assay versionDiluted DNA std. (µl)1X TE buffer (µl)Final DNA amount (ng)2High-range (1-200 ng µl-1)10002003Use 2 ng μl-1 standard50501004
1090205
19916
010007Low-range (50 pg µl-1 - 5 ng µl-1)100058Use 0.05 ng μl-1 standard50502.59
10900.510
1990.0511
01000
?. [Prepare reaction]
Pipette 99 µl of 1X TE buffer in the remaining wells.
[1X TE buffer]
Tip: use a mechanical or electronic dispenser during this step and step no. 9 to speed up the work.
?. [Prepare reaction]
Pipette 1 µl of the unknown DNA samples in the remaining wells.
[of DNA sample]
Use either a diluted sample in case the concentration is expected to be higher than the dynamic range limit or larger volume in case the concentration is expected to be below the detection limit.
?. [Prepare reaction]
Pipette 100 µL of the PicoGreen® work solution in each well, including the standard and unknown sample wells.
[PicoGreen work solution]
?. [Prepare reaction]
Protect the 96-well plate from light and incubate for 2-5 min at room temperature.
?. [Measure samples]
Place the plate in a plate reader and measure the fluorescence according to the following parameters:Excitation                   ~480 nmEmission                    ~520 nmIntegration time            40 sLag time                         0 sGain                                OptimalNumber of flashes      10Calculated well             highest standardShaking                          5 s
It is also possible to set the gain to a fixed value (e.g. 100). If the fluorescence values of the standard drop over time this could indicate damage to the reagents or the DNA standard.
?. [Measure samples]
Plot the measured fluorescent values of the standard samples against their known concentrations and fit a linear curve using linear regression. Make sure that the coefficient of determination (R2) is close to 1 (typically > 0.99). Calculate the DNA concentrations in the unknown samples using the slope and intercept parameters of the linear equation. Output values you obtained are in ng µl-1, assuming 1 µl of each sample was used.  See attached example spreadsheet.
Do not forget to account for any dilutions when calculating the concentration of the DNA in the unknown samples.