Mar 05, 2019
Quanti-iT™ Pico Green dsDNA Assay (Invitrogen P7589)
- Matthew Sullivan Lab1
- 1Matthew Sullivan Lab, University of Arizona/Ohio State University
- Earth Microbiome Project
External link: https://doi.org/10.1371/journal.pone.0212355
Protocol Citation: Matthew Sullivan Lab 2019. Quanti-iT™ Pico Green dsDNA Assay (Invitrogen P7589). protocols.io https://dx.doi.org/10.17504/protocols.io.c5zy75
Manuscript citation:
Ul-Hasan S, Bowers RM, Figueroa-Montiel A, Licea-Navarro AF, Beman JM, Woyke T, Nobile CJ (2019) Community ecology across bacteria, archaea and microbial eukaryotes in the sediment and seawater of coastal Puerto Nuevo, Baja California. PLoS ONE 14(2): e0212355. doi: 10.1371/journal.pone.0212355
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
Created: June 23, 2015
Last Modified: March 05, 2019
Protocol Integer ID: 921
Guidelines
| DNA Standard | Vol.μl DNA | Vol.μl 1x TE | Vol μl PicoGreen (1:200) | Final DNA Standard ng/mL | |
| Low DNA Standard | 0 | 100 | 100 | 0 | |
| (1:1000 of 100 μg/ml) | 1 | 99 | 100 | 0.5 | |
| 5 | 95 | 100 | 2.5 | ||
| 10 | 90 | 100 | 5.0 | ||
| 20 | 80 | 100 | 10 | ||
| 50 | 50 | 100 | 25 | ||
| 80 | 20 | 100 | 40 | ||
| 100 | 0 | 100 | 50 | ||
| High DNA Standard | 0 | 100 | 100 | 0 | |
| (1:50 of 100 μg/ml) | 1 | 99 | 100 | 10 | |
| 5 | 95 | 100 | 50 | ||
| 10 | 90 | 100 | 100 | ||
| 20 | 80 | 100 | 200 | ||
| 50 | 50 | 100 | 500 | ||
| 80 | 20 | 100 | 800 | ||
| 100 | 0 | 100 | 1000 |
Materials
MATERIALS
Quant-it™ PicoGreen® dsDNA Assay KitLife TechnologiesCatalog #P7589
STEP MATERIALS
Quant-iT PicoGreen dsDNA kitThermo ScientificCatalog #P7589
Quant-iT PicoGreen dsDNA kitThermo ScientificCatalog #P7589
Protocol materials
Quant-iT PicoGreen dsDNA kitThermo ScientificCatalog #P7589
Quant-it™ PicoGreen® dsDNA Assay KitLife TechnologiesCatalog #P7589
Quant-iT PicoGreen dsDNA kitThermo ScientificCatalog #P7589
Quant-iT PicoGreen dsDNA kitThermo ScientificCatalog #P7589
Before start
Determine number of samples and standards to test in 96 well plate format. Multiply by 2 if running everything in duplicate for total number of wells.
Use a black-walled plate with black bottoms if possible. Black-sided wells with clear bottoms or white-sided wells will also work, but background will be higher due to reflected fluorescence in the wells. Do not use clear microtiter plates for fluorescence readings.
You will need 100 μl diluted PicoGreen reagent per well.
Total amount of 1X TE per assay will be 200 μl per well which includes the amount of TE used to dilute the PicoGreen reagent.
Pico Green dsDNA Assay
Pico Green dsDNA Assay
Warm Quant-iT PicoGreen reagent to room temp in the dark.
Note
PicoGreen reagent is diluted in dimethylsulfoxide (DMSO) which solidifies at refrigerator temperatures. It must be completely liquified before use by allowing it to come to room temperature. Vortex solution briefly to mix well and centrifuge for 5 sec to bring liquid to bottom of tube; then dispense for use in the assay. PicoGreen reagent is also light-sensitive, so reagent should be protected from light.
Quant-iT PicoGreen dsDNA kitThermo ScientificCatalog #P7589
Prepare 1XTE buffer from 20X stock solution using nuclease-free water: will need 200 μl/well (for diluting standards, samples and PicoGreen).
Note
Prepare 1X TE by pipetting 2.5 mL of 20X stock TE into a sterile 50 mL centrifuge tube and filling to 50 mL mark with molecular biology grade water. Invert tube to mix.
Dilute DNA standard to either “High” 2 μg/mL (1:50 of λ DNA stock) or “Low” 50 ng/mL (1:1000 of λ DNA stock).
Note
It is best to run standards in duplicate, and if amount of DNA in samples is unknown or varys widely, it is also best to run both the high and low DNA standards.
Determine amount of sample to assay (eg, 2μl sample in total of 100μl TE buffer). Add correct amount of TE buffer to all wells. Add standards to wells. Then add samples to wells.
Note
See Guidelines for amount of DNA standards to add to standard wells.
Dilute PicoGreen 1:200 in TE buffer and protect from light until ready to add to plate.
Note
A 1:200 dilution of PicoGreen reagent is prepared by adding 10 μl of PicoGreen per 2 mL of 1X TE buffer. You will need 100 ul diluted PicoGreen per well containing 100 ul sample.
Add equivalent volume (100 μl) of diluted PicoGreen to every well (keeping plate in the dark as much as possible).
Tap plate to mix.
Incubate 5 minutes at room temperature keeping plate in the dark.
00:05:00
Take fluorescent readings using 485nm excitation and 535nm emission filters.
Determine standard curve and calculate concentration of DNA in samples (see table in the guidelines).