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DNA quantification using absorbance (A260) and fluorescent methods (Qubit™ and Quant-iT™/PicoGreen™)

Andrea Krumm BMG LABTECH GmbH, 77799 Ortenberg, Germany 10/2020
  • BMG LABTECH multi-mode plate readers are compatible with common DNA quantification methods
  • Quantification of double-stranded DNA with hundreds of samples in parallel
  • Detect down to 0.8 pg double-stranded DNA per well

Table of contents

Introduction

Double-stranded DNA not only holds all information to build a human but is also a popular molecule to study. Whether expression levels or infections that are measured by PCR, or next generation sequencing to generate sequence information and to detect mutations responsible for disease. DNA-based methods require the quantification of DNA: here we present commonly used methods based on absorbance or fluorescence to quantify DNA at all concentrations, volumes and in any throughput.


Materials & Methods

  • 384 well UV-transparent (Greiner #781801)
  • black 384 well small volume (Greiner #784076)
  • LVis Plate (BMG LABTECH)
  • CLARIOstar® Plus, FLUOstar Omega (BMG LABTECH)
  • Qubit™ dsDNA BR (ThermoFisher #Q32850)
  • Qubit™ dsDNA HS (ThermoFisher #Q32854)
  • Quant-iT™ Picogreen™ (ThermoFisher #P11469)
  • Lambda DNA (ThermoFisher #SD0011)
  • Roti® Stock 100x TE and H2O (Carl Roth)

 

Experimental procedure 
Lambda DNA was diluted in 1x TE buffer to concentrations ranging between 1 pg/ml and 130 µg/ml. Qubit BR and Qubit HS assays were performed by diluting DNA 1:10 in the working solution. For 96 well plates, 20 µl DNA were mixed with 180 µl of dye solution, for 384 well plates, 2 µl of DNA were mixed with 18 µl of dye solution. In the Quant-iT/PicoGreen assay, DNA and working solution were mixed 1:1. A 96 plate well was filled with 100 µl of dye and 100 µl of DNA, a 384 well with 10 µl of each solution. Absorbance-based measurements employ the DNA as it is and volumes of 100 µl, 50 µl and 2 µl were placed into a 96 well, 384 well or LVis plate, respectively.


All measurements were done in triplicates and 12 replicates for blank measurements.

 

Instrument settings

 

Absorbance (predefined protocol dsDNA)
Optic settings Absorbance, endpoint
Spectrum 1nm resolution  220-360 nm
General settings Number of flashes 22
Settling time 0.2 s (384)
Settling time 0.5 s (96)

 

 

Fluorescence intensity, CLARIOstar Plus
Optic settings Absorbance, endpoint
Monochromator FITC default
Excitation 483-14
Auto dichroic
Emission 530-30
General settings Number of flashes 20
Settling time 0.2 s
Start settings Focus: Autofocus
Dynamic Range: EDR

 

Fluorescence intensity, FLUOstar Omega
Optic settings Absorbance, endpoint
Filters Excitation: 485
Emission: 520
General settings Number of flashes 20
Settling time 0.2 s
Start settings Gain adjustment On highest standard, target value 90%

 

 

Results & Discussion

Absorbance-based dsDNA quantification BMG LABTECH multi-mode microplate readers acquire absorbance spectra using an ultrafast UV/vis spectrometer. DNA spectra can be measured in volumes from 2-100 µl using different plate formats. The resulting absorbance spectra were automatically analyzed with the MARS analysis software. Next to DNA concentration, it calculates the A260/A280 ratio to determine protein contaminations (Fig 1).

Fluorescence-based DNA quantification
Fluorescent dyes for the quantification of DNA are used because, in contrast to absorption, they are more specific and detect lower DNA concentrations. Here, we compared three commonly used dyes: Qubit high sensitivity and low sensitivity kits and the Quant-iT PicoGreen dsDNA quantification kit. All kits were measured according to the instructions of use and with the same standards.


Upon detection, the data were evaluated with the MARS analysis software. It calculated the concentration of unknowns, measured in parallel to the standard curve. The results and concentration ranges of the three assays are compared in Figure 3. It shows the Quant-iT and  Qubit HS kits covering a large concentration range and being sensitive for low DNA concentrations. The Qubit BR kit covers high DNA concentrations.

Comparison of plate formats and reader types
Further, the limit of detection was calculated for all three assay kits and the absorbance method. The detection limit is the concentration at the blank value plus three times the standard deviation of 12 blanks (mean blanks + 3*SDblank). The results are shown in Table 1 and figure 4. The maximal measurable concentrations for fluorescent dyes are taken from the kits specifications.

Table 1. Comparison of microplate-based DNA quantification methods regarding sample volume and minimum DNA input (for CLARIOstar Plus)

 

MethodPlateSample VolumeMinimum DNA input
A260LVis2 µl5000 pg
A26096 well100 µl33000 pg
A260384 well50 µl 10500 pg
QubitHS96 well1-20 µl3.4 pg
QubitHS384 well sv0.1-2 µl0.8 pg
Qubit BR96 well1-20 µl 70 pg
Qubit BR384 well sv0.1-2 µl13.6 pg
Quant-iT/ PicoGreen96 well100 µl3.5 pg
Quant-iT/ PicoGreen384 well sv10 µl2.3 pg

 

 

Conclusion

BMG LABTECH microplate readers reliably detect double-stranded DNA. The instruments cover quantification of any sample concentration and volume. The data shown above help to find the right instrument and quantification method for your needs.

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