Streamlining plasmid DNA analysis from bacterial cultures

A wide range of scientific laboratories rely on DNA purification and quantitation applications. Plasmid DNA preps and their subsequent quantification are key to the success of workflows across a vast range of applications, from cloning and PCR to transfection and sequencing.

This article explores the use of a versatile plasmid DNA purification method with relatively small volumes of bacterial culture, for instance, those grown in a 96-well microplate format.

This assay produces concentrations of DNA preps that can be easily assessed using a sensitive fluorescent assay read on the SpectraMax^® iD3s Multi-Mode Microplate Reader from Molecular Devices.

This method offers a number of benefits, including:

• Sensitive fluorescent quantitation of DNA down to 50 pg/mL, meaning it is well-suited for use with plasmid preps from bacteria grown in microplates
• A powerful linear dynamic range spanning over four orders of magnitude
• Easy results analysis via a range of ready-to-use protocols in the SoftMax^® Pro software

Materials

• pGEM^®-T Vector Systems with JM109 Competent Cells (Promega cat. #A3610)
• 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)
• SpectraMax iD3s Multi-Mode Microplate Reader (Molecular Devices)

The SpectraMax iD5e Multi-Mode Microplate Reader also offers fluorescence detection, yielding equivalent results for the PicoGreen assay.

Methods

Bacterial culture preparation and cell harvest

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

A SpectraMax iD3s Multi-Mode Microplate Reader was used to monitor culture growth for 18 hours, with continuous shaking between reads and absorbance measurements completed every 15 minutes.¹

After incubation had been completed, 20 samples (200 µL each) of the bacterial cultures were transferred to microcentrifuge tubes before being pelleted via centrifugation at 6000 RCF (Eppendorf Minispin Plus Microcentrifuge) for one minute.

Supernatants were discarded before extracting plasmid DNA using the Mag-Bind UltraPure Plasmid DNA kit. Extraction was performed according to the manufacturer’s product manual.

Plasmid DNA extraction steps

• Bacterial pellets were pipetted up and down to fully resuspend them in the provided resuspension buffer (containing RNase A). This process continued until no clumps remained.
• Lysis buffer was added, with tubes gently inverted between six and eight times to ensure they were mixed. Lysates were briefly incubated at room temperature until they became viscous and clear.
• Neutralization buffer was added, with tubes inverted immediately and thoroughly to precipitate genomic DNA and cell debris.
• Lysates were clarified via centrifugation, and supernatants were then transferred to a 96-well deep-well plate.
• Binding buffer and Mag-Bind paramagnetic beads were added to the cleared lysates.
• The mixtures were gently mixed via pipetting, ensuring that plasmid DNA was able to bind to the bead surface.
• The 96-well deep-well plate was placed on a magnetic separation rack to allow the solution to clear before the supernatants were discarded while ensuring the beads were not disturbed.
• Beads were washed sequentially with the provided wash buffers to remove contaminants such as salts and proteins.
• Beads were air-dried briefly on the magnetic rack. After the final wash, residual ethanol was removed.
• Low-salt elution buffer was used to elute plasmid DNA from the beads. Next, 100-µL eluates were collected into a fresh 96-well plate following magnetic separation.

Quant-iT PicoGreen dsDNA assay

The method for this assay was completed in accordance with 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. DNase-free water was used to dilute the concentrated buffer supplied with the kit 20-fold.
• An aqueous working solution of Quant-iT PicoGreen reagent was prepared by diluting the concentrated DMSO solution 200-fold in the earlier prepared 1X TE buffer. It was important to use this solution within a few hours of its preparation.
• A 2 µg/mL stock solution of the kit’s lambda DNA standard was prepared for the DNA standard curve by diluting it 50-fold in TE.
  • A series of standards ranging from 50 pg/mL to 1000 ng/mL was set up for this application. This was done in a 1:3 dilution series.
• Next, standards were pipetted into a solid-black 96-well microplate (assay plate) in triplicate at 100 µL per well. This included a set of buffer blank wells containing TE only (no DNA).
• Prepared plasmid DNA samples were diluted 1:20 in TE, then 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 added to each individual assay well. A plate shaker was used to mix the plate briefly before it was incubated for two to five minutes at room temperature while being protected from light.

Results

One of the SoftMax Pro software’s Protocol Library’s ready-to-use assay protocols was used to generate data on a SpectraMax iD3s reader. This was done with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Automatic PMT Gain was used, with a 400 ms integration time.

This protocol included optimized settings for the PicoGreen assay. The microplate was read from the top using the optimal read height of 2.37 mm, with this read height determined using the read height optimization feature within the software.

The template editor included in the assay protocol includes pre-set groups. These groups were used to designate wells as samples or standards, after which the replicate standard deviation, DNA concentrations, and %CV were automatically calculated. A sample dilution factor of 20 was applied to reach each sample’s final DNA concentration.

Plasmid DNA prep concentrations were quantitated from the DNA standard curve, which was set up as described in Figure 1. Concentrations of these preps ranged from 134.8 ng/mL to 993.8 ng/mL (Table 1). These were calculated from sample RFU values, and all values 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 displays sample IDs, RFU values, calculated concentrations, replicate statistics, and final adjusted concentrations. Source: Molecular Devices UK Ltd

Conclusion

The easy-to-use PicoGreen DNA quantitation assay boasts straightforward reagent preparation and assay setup. The SoftMax Pro software’s data generation efficiency complements this assay effectively.

Accurate concentrations can be calculated from a standard curve, with the software also offering a clear display of the results and their precision. This is achievable via a protocol available in the software, which can be easily adapted to a user’s particular assay setup.

The combination of the SpectraMax iD3s reader and the assay’s sensitivity enables accurate quantitation of plasmid preps from small-volume cultures grown in a microplate.

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 bioanalytical measurement systems, software and consumables for life science research, pharmaceutical and biotherapeutic development. Included within a broad product portfolio are platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. These leading-edge products enable scientists to improve productivity and effectiveness, ultimately accelerating research and the discovery of new therapeutics. Molecular Devices is committed to the continual development of innovative solutions for life science applications. The company is headquartered in Silicon Valley, California, with offices around the globe. For more information, please visit www.moleculardevices.com.

Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.net, which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.