Reliable Plasmid DNA Quantitation From Bacterial Microplates

DNA purification and quantitation are among the most widely performed applications in scientific laboratories. In this context, plasmid DNA preps and their subsequent quantification are critical to the success of workflows across a diverse array of applications, from cloning and PCR to transfection and sequencing.

This article discusses a versatile plasmid DNA purification method suitable for use with relatively small volumes of bacterial culture, for example, those grown in a 96-well microplate format. The DNA prep concentrations produced by this assay are easily evaluated via a sensitive fluorescent assay read on Molecular Devices’ SpectraMax^® iD3s Multi-Mode Microplate Reader.

This method offers a range of advantages, including:

• Sensitive fluorescent quantitation of DNA down to 50 pg/mL, making it suitable for plasmid preps from microplate-grown bacteria
• A linear dynamic range spanning more than four orders of magnitude
• Easy analysis of results via the SoftMax^® Pro software’s ready-to-use protocols

Materials

• Quant-iT^™ PicoGreen^™ dsDNA Assay Kit (Thermo Fisher Scientific cat. #P7589)
• 96-well solid black microplate (Greiner Bio-One cat. #655076)
• Mag-Bind^® Ultra-Pure Plasmid DNA 96 Kit (Omega Bio-tek cat. #M1258-01)
• pGEM^®-T Vector Systems with JM109 Competent Cells (Promega cat. #A3610)
• SpectraMax iD3s Multi-Mode Microplate Reader (Molecular Devices)

The SpectraMax iD5e Multi-Mode Microplate Reader yields equivalent results for the PicoGreen assay and also features fluorescence detection.

Methods

Bacterial Culture Preparation and Cell Harvest

Escherichia coli containing the target plasmid (JM109 strain transformed with pGemT vector + control insert) was cultured overnight. This was performed in a 96-well plate containing LB broth supplemented with 25 μg/mL ampicillin at 37 °C.

Culture growth was monitored for 18 hours using a SpectraMax iD3s Multi-Mode Microplate Reader. Absorbance measurements were performed every 15 minutes, with continuous shaking between readings.¹

A total of 20 samples (200 µL each) of the bacterial cultures were transferred to microcentrifuge tubes following incubation. These were then pelleted via centrifugation at 6000 RCF (Eppendorf Minispin Plus Microcentrifuge) for one minute.

The supernatants were then discarded, with plasmid DNA extracted via the Mag-Bind UltraPure Plasmid DNA kit in line with the product manual from the manufacturer.

Plasmid DNA Extraction Steps

• Bacterial pellets were totally resuspended in the provided resuspension buffer (containing RNase A). This was done by pipetting up and down until no clumps remained.
• Lysis buffer was added gently before the tubes were inverted six to eight times to mix them. Lysates were incubated briefly at room temperature until they became viscous and clear.
• Neutralization buffer was added, with the tubes immediately and thoroughly inverted to precipitate cell debris and genomic DNA.
• Lysates were clarified via centrifugation before transferring the supernatants to a 96-well deep-well plate.
• Mag-Bind paramagnetic beads and binding buffer were each added to the newly cleared lysates.
• The mixtures were gently pipetted to mix them and allow plasmid DNA to bind to the bead surface.
• The 96-well deep-well plate was placed on a magnetic separation rack until the solution cleared. Supernatants could then be discarded without disturbing the beads.
• Beads were sequentially washed with the provided wash buffers to remove salts, proteins, and other contaminants.
• Following the final wash, beads were briefly air-dried on the magnetic rack to remove residual ethanol.
• Plasmid DNA was eluted from the beads with low-salt elution buffer. After magnetic separation, 100-µL eluates were collected into a fresh 96-well plate.

Quant-iT PicoGreen dsDNA Assay

This assay’s method followed the instructions in the Quant-iT PicoGreen dsDNA Reagent and Kit User Guide. A 200 µL assay volume was used to fit the 96-well microplate format.

• 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was prepared by diluting the concentrated buffer supplied with the kit 20-fold using DNase-free water.
• An aqueous working solution of Quant-iT PicoGreen reagent was prepared by creating a 200-fold dilution of the concentrated DMSO solution in the pre-prepared 1X TE buffer. This solution was used within a few hours of its preparation.
• A 2 µg/mL stock solution of the lambda DNA standard provided with the kit was prepared for the DNA standard curve. This was achieved by diluting it 50-fold in TE.
  • A series of standards ranging from 50 pg/mL to 1000 ng/mL was set up in a 1:3 dilution series for the purposes of this application.
• Standards were then pipetted into a solid-black 96-well microplate (assay plate) at 100 µL per well. This was done in triplicate, including a set of buffer-blank wells containing only TE (no DNA).
• Existing plasmid DNA samples were diluted 1:20 in TE. Next, 100 µL of diluted sample was pipetted into assay plate wells in duplicate.
• A total of 100 µL of aqueous working solution of Quant-iT PicoGreen reagent was inserted into each assay well. Next, the plate was briefly mixed with a plate shaker, then protected from light and incubated for two to five minutes at room temperature.

Results

The Protocol Library of SoftMax Pro software offers a range of ready-to-use assay protocols. One of these protocols was used to generate data on a SpectraMax iD3s reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. PMT Gain was set to automatic in this instance, with an integration time of 400 ms.

The protocol includes optimized settings for the PicoGreen assay. An optimal read height of 2.37 mm was set, with the microplate read from the top. This read height was determined using the software’s Read Height Optimization feature.

The assay protocol’s Template Editor includes preset groups, and this was used to designate wells as either standard or sample. DNA concentrations, replicate standard deviation, and %CV were all calculated automatically. The final DNA concentration for each sample was achieved using a sample dilution factor of 20.

Plasmid DNA prep concentrations were quantitated from the DNA standard curve. Figure 1 shows the setup of this curve. Prep concentrations ranged from 134.8 ng/mL to 993.8 ng/mL (Table 1); these were calculated from sample RFU values, and all fell within the range covered by the DNA standards.

Figure 1. DNA standard curve, with standard concentrations ranging from 50 pg/mL to 1000 ng/mL (r² = 0.998). From this standard curve, concentrations of plasmid DNA prep samples were interpolated. Image Credit: Molecular Devices UK Ltd

Table 1. Plasmid prep sample DNA concentrations. A group table in SoftMax Pro software displays sample IDs, RFU values, and calculated concentrations, including replicate statistics and final adjusted concentrations. Source: Molecular Devices UK Ltd

Conclusion

The PicoGreen DNA quantitation assay is easy to use and streamlined, from reagent preparation to assay setup. This assay is well supported by the SoftMax Pro software’s efficiency in generating data.

The software’s protocols enable accurate concentration calculation from a standard curve and clearly display the results and their precision. This protocol can be readily adapted to a user’s specific assay setup.

The assay’s sensitivity and the efficiency of the SpectraMax iD3s reader ensure accurate quantitation of plasmid preps from microplate-grown small-volume cultures.

References and Further Reading

1. Monitor effects of nutrient or antibiotic addition on bacterial growth. Molecular Devices application note 2827A (2025).
2. Sensitive fluorescent quantitation of DNA with the Quant-iT PicoGreen dsDNA Assay Kit. Molecular Devices application note 2373B (2022).

Acknowledgments

Produced from materials originally authored by Sushmita Sudarshan, PhD, and Cathy Olsen, PhD, from Molecular Devices.

About Molecular Devices UK Ltd

Molecular Devices is one of the world’s leading providers of high-performance life science technology. We make advanced scientific discovery possible for academia, pharma, and biotech customers with platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. From cancer to COVID-19, we've contributed to scientific breakthroughs described in over 230,000 peer-reviewed publications.

Over 160,000 of our innovative solutions are incorporated into laboratories worldwide, enabling scientists to improve productivity and effectiveness – ultimately accelerating research and the development of new therapeutics. Molecular Devices is headquartered in Silicon Valley, Calif., with best-in-class teams around the globe. Over 1,000 associates are guided by our diverse leadership team and female president who prioritize a culture of collaboration, engagement, diversity, and inclusion.

To learn more about how Molecular Devices helps fast-track scientific discovery, visit www.moleculardevices.com.

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