Nov 29, 2021
Version 3
Quant-iT™ PicoGreen® dsDNA Quantification V.3
- 1Soil and Water Research Inrastructure
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: eva.petrova@bc.cas.cz
External link: https://www.thermofisher.com/order/catalog/product/P11496
Protocol Citation: Roey Angel, Eva Petrova 2021. Quant-iT™ PicoGreen® dsDNA Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.b2etqbenVersion created by Roey Angel
Manuscript citation:
Angel, R., Claus, P., and Conrad, R. (2012). Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions. ISME J 6, 847–862. doi:10.1038/ismej.2011.141.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 29, 2021
Last Modified: November 29, 2021
Protocol Integer ID: 55475
Keywords: DNA, quantification, nucleic acids, fluorometric assay, high-throughput,
Abstract
The following protocol is intended for the quantification of double-stranded DNA using Quant-iT™PicoGreen® dsDNA Assay Kit (ThermoFisher). This protocol is a simplified and condensed version of the full protocol from the manufacturer. The procedure described here is for 96 reactions. If samples are run in duplicates, then this should allow quantifying 40 samples.
Attachments
mp07581.pdf
240KB
Materials
MATERIALS
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
STEP MATERIALS
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
Protocol materials
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
Safety warnings
Quant-iT™ PicoGreen® reagent is classified as Not Hazardous. Nevertheless, the user should always consult the MSDS accompanying any of the reagents and apparatus described in this protocol.
Before start
- This protocol is optimised for measuring an entire 96-well plate. It assumes that 16 wells will be used for measuring the standards and the blank samples (in duplicates) and 80 wells will be used for measuring unknown DNA samples (typically in duplicates).
- The protocol can be easily adjusted for a lower number of samples by reducing the volume of the working solutions of the reagents. Note though that enough TE should be retained for diluting the standard stock solution (490 or 680 µl), for potentially diluting the unknown samples, if their concentration is too high, and for accounting for pipetting errors. To fill the plate, 19.2 ml of TE is needed. So if only 40 wells are to be used for measuring unknown samples prepare about ml of TE buffer.
- The dynamic range of the assay is between 50 pg ml-1 to 1000 ng ml-1. This translates into DNA sample concentrations of 0.05-5 ng µl-1 and 1-200 ng µl-1 in the low-range and high-range assays, respectively. Samples with higher DNA concentration need to be diluted (e.g. in DNase-free water or TE buffer).
- Note that some compounds that can be present as DNA contaminations (e.g. salts, ethanol, detergents, proteins) are claimed by the manufacturer to not interfere with the measurement. Please refer to the full protocol for a list of these compounds and their effect on the measurement. Also, equimolar presence of ssDNA and RNA in the sample should have only minimal effect on the quantitation results.
Prepare reaction
Prepare reaction
53m
53m
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.
Note
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.
Note
Quant-iT™PicoGreen® dsDNA reagent is light sensitive and should be protected from light at all times.
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
20m
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.
1.1 µL 20X TE buffer
20.9 µL nuclease-free water
2m
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.
10 µL λ DNA-standard stock solution
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.
5 µL diluted DNA-standard solution
195 µL 1X TE buffer
2m
If needed, prepare a dilution of each sample in 1X TE buffer so that the reading will be within the dynamic range.
Note
It is advisable to run samples in duplicates for a more accurate quantification
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.
9950 µL 1X TE buffer
50 µL PicoGreen®
2m
Prepare the following standard mixture in the first two columns of the black, sterile, 96-well plate:
| Assay version | Diluted DNA std. (µl) | 1X TE buffer (µl) | Final DNA amount (ng) | |
| High-range (1-200 ng µl-1) | 100 | 0 | 200 | |
| Use 2 ng μl-1 standard | 50 | 50 | 100 | |
| 10 | 90 | 20 | ||
| 1 | 99 | 1 | ||
| 0 | 100 | 0 | ||
| Low-range (50 pg µl-1 - 5 ng µl-1) | 100 | 0 | 5 | |
| Use 0.05 ng μl-1 standard | 50 | 50 | 2.5 | |
| 10 | 90 | 0.5 | ||
| 1 | 99 | 0.05 | ||
| 0 | 100 | 0 |
Equipment
96-well microtiter plate
NAME
Nunc
BRAND
265301
SKU
LINK
black, flat bottom
SPECIFICATIONS
10m
Pipette 99 µl of 1X TE buffer in the remaining wells.
99 µL 1X TE buffer
Note
Tip: use a mechanical or electronic dispenser during this step and step no. 9 to speed up the work.
Equipment
Multipette E3
NAME
Eppendorf
BRAND
4987000010
SKU
LINK
electronic dispenser
SPECIFICATIONS
5m
Pipette 1 µl of the unknown DNA samples in the remaining wells.
1 µL of DNA sample
Note
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.
10m
Pipette 100 µl of the PicoGreen® work solution in each well, including the standard and unknown sample wells.
100 µL PicoGreen work solution
2m
Protect the 96-well plate from light and incubate for 2-5 min at room temperature.
00:02:00
5m
Measure samples
Measure samples
5m
5m
Place the plate in a plate reader and measure the fluorescence according to the following parameters:
Excitation ~480 nm
Emission ~520 nm
Integration time 40 s
Lag time 0 s
Gain Optimal
Number of flashes 10
Calculated well highest standard
Shaking 5 s
Note
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.
Equipment
Synergy 2
NAME
absorbance microplate reader
TYPE
BioTek
BRAND
Synergy2
SKU
LINK
5m
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.
Note
Do not forget to account for any dilutions when calculating the concentration of the DNA in the unknown samples.
PicoGreen_example.xlsx 10m