KS USDA, St. Amand Revised on 5-July-2017
Quantification of DNA Using PicoGreen, Biomek, Mantis, and Omega reader
Preparation of Samples for Standard Curve
To standardize the DNA concentrations we must prepare a dilution of known concentrations. This is done using the λ-DNA supplied with the PicoGreen reagent. The λ-DNA standards may be saved at 4 °C and used for up to 1 month, but new standards usually works best.
In one 8 tube strip, prepare λ-DNA (100ng/ul) by vortexing and centrifuging and following the table below. Write the date on the strip.
Well Number / (Low Vol) Microliters of λ-DNA:TRIS (total vol. varies) / STD λ-DNA conc. (ng/µL)1 / 10:0 / 100
2 / 8:2 / 80
3 / 6:4 / 60
4 / 6:9 / 40
5 / 5:20 / 20
6 / 5:45 / 10
7 / 5:95 / 5
8 / 0:50 / 0
Preparation of PicoGreen-TE
Prepare a PicoGreen-TE in a foil covered bottle. Use the PicoGreen reagent at dilution of 200X. PicoGreen must be protected from light.
For each DNA plate to be quantified add:
· 19.9 ml 1x TE
· 100 µL PicoGreen
Protect from light and mix by swirling bottle or spinbar. (An additional 7.960 mL TE with 40µL PicoGreen is needed for the standards and overage.)
Preparation of λ-DNA Standards
In a new Costar (or Brand) black plate:
· Add 200 µL 1x PicoGreen-TE to 8 wells of a column on the plate.
· Vortex, spin, and transfer 2µL λ-DNA from the 8 tube strip using a precision 2 µL pipetter.
· Use Biomek script to mix the standards or mix 10x by hand using 200 ul volume.
· Set the standards aside in the dark.
Preparation of Samples
DNA samples in plate format. Prepare DNA plate by thawing, vortexing, and spinning down.
In a new Costar (or Brand) black plate:
· For each sample plate, use the Biomek script to pool samples with PicoGreen-TE.
· Add PicoGreen-TE to reservoir, black plate, p50 sample tips, p200 PicoGreen-TE tips, and DNA to robot deck.
· The script will add 200 µL of PicoGreen-TE and 2 ul of sample to each well and mix 12X.
· Cover the plates and protect from light.
Read Florescence of Plates using the Omega reader
DNA samples must incubate at room temperature for at least 5 minutes prior to reading. Protect samples from light.
Using the Omega reader:
· Turn on the Omega, open the Omega software, select the PicoGreen script button.
· Insure that the following settings are correct: Excitation = 485, Emission = 520, Gain = 800, and that the optics are set to 6 mm.
· Name the plate in the ID1 field, press run, and the plate will be read. Read the standard plate and all sample plates the same way.
· Output files will be in the "Data" folder on the desktop. Copy all files Dolly or a USB disk. The files in the "Data" folder are erased often. Backup your data.
Use the Excel Calculator to Convert Florescence to DNA Concentration
Get the "PicoGreen-Mantis Calculator" from our website.
Using the Calculator:
· Paste the standard data and the sample data into the appropriate locations using the "Paste Values" option.
· Adjust the desired final concentration, desired maximum volume, and current well volume in the blue fields. Do not change any other locations of the calculator.
· Check that no wells have exceeded the maximum volume.
· The sheet will calculate the current (stock) concentrations in ng/ul, the amount of TRIS (or water) to add to get the desired concentration, and the final concentrations after dilution.
· Copy the Mantis volumes into another file and use the Mantis to dilute the original plate or an aliquot from the original plate.