Nov 20, 2025
A Quantitative Determination of Dielectric Thresholds for DNA Precipitation by Ethanol
- Agam Vohra1,2
- 1University Health Network;
- 2Princess Margaret Cancer Centre
Protocol Citation: Agam Vohra 2025. A Quantitative Determination of Dielectric Thresholds for DNA Precipitation by Ethanol. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqp8q5vk5/v1
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
This protocol provides a quantitative, dielectric-threshold model for ethanol-mediated DNA precipitation, validated experimentally across four DNA concentrations. All steps have been optimized for reproducibility, minimal reagent waste, and compatibility with high-throughput molecular workflows.
Created: November 17, 2025
Last Modified: November 20, 2025
Protocol Integer ID: 233066
Keywords: DNA precipitation, Dielectric constant, Ethanol precipitation, Beer–Lambert Law, Coulombic interactions, Spectrophotometry, DNA recovery, Biophysical modelling, Nucleic acid purification, ethanol volumes during dna precipitation, dna precipitation by ethanol current dna preservation practice, ethanol current dna preservation practice, ethanol concentration, dna concentration, minimum ethanol requirement for human, predicting ethanol volume, dielectric constants of ethanol, dna precipitation, somatic dna precipitation, ethanol requirement, standard dna precipitation protocol, minimum ethanol requirement, ethanol volume, dna molarity, dna precipitate, ethanol, quantitative determination of dielectric threshold, dielectric threshold, spectrophotometric absorbance data, different dna condition, consistent dielectric threshold, compromising dna integrity, dna integrity, precipitation profile, dielectric constant, reagent waste
Abstract
Current DNA preservation practices rely on empirically determined volumetric rules rather than quantitative theoretical frameworks. Existing models overestimate ethanol requirements, limiting reproducibility and hindering the development of efficient protocols. A dielectric threshold–based model for predicting ethanol volumes during DNA precipitation establishes a framework that will reduce reagent waste and thus, environmental impact. This quantitative framework predicts the minimum ethanol requirement for human somatic DNA precipitation as a function of DNA molarity, derived from spectrophotometric absorbance data using the Beer-Lambert Law and Coulomb’s Law. For four different DNA conditions, 12 replicate samples were prepared across an ethanol concentration gradient from 54% to 90% v/v in 2% increments (n = 12 per concentration). DNA molarity and the dielectric constants of ethanol (ε = 24.5) and water (ε = 80.1) were used to calculate the dielectric constant of each solution, enabling determination of the threshold for DNA precipitation. DNA precipitation begins at 58-60% ethanol and reaches a maximum yield of 95% at 72% ethanol. Across all four DNA concentrations (2, 5, 8, and 10 ng/µL), replicates demonstrated consistent dielectric thresholds and precipitation profiles, generating a reproducible sigmoidal recovery curve. ε = 40.07 is the dielectric threshold at which DNA precipitates optimally, corresponding to the point at which our yield plateaus (varying ± 3% across replicates). These results validate the dielectric-threshold framework, showing a slight shift in the onset relative to theoretical predictions, while demonstrating that ethanol volumes can be optimized without compromising DNA integrity. Adopting the dielectric-threshold framework reduces ethanol requirements by 3% compared to standard DNA precipitation protocols, while ensuring optimal yield and quality. Standardizing this model will improve reproducibility, enable automation, and reduce biohazardous and flammable waste across high-throughput workflows.
Guidelines
Controls
Three controls are utilized in this protocol to verify that DNA precipitation is a function of ethanol dielectric effects:
Negative Control: A tube containing nuclease-free water, sodium acetate, and glycogen, without DNA, in the same concentrations as the other samples, was mixed with all variations in ethanol concentrations across the gradient.
Positive Control: A 20ng/µL sample of DNA with sodium acetate and glycogen was precipitated at 70% to confirm that precipitation will proceed as expected beyond the threshold under standard conditions.
Spectrophotometer Blank: The spectrophotometer was blanked with a solution containing the base amounts of sodium acetate, glycogen, and nuclease-free water for all A260 baseline samples.
Materials
- 96-well plate x 3
- 1mL or 1.5mL microcentrifuge or 15mL centrifuge tubes x 500
- Centrifuge
- Incubator
- Spectrophotometer
- Gloves (nitrile or neoprene) are necessary to resist ethanol corrosion and prevent contamination.
- Lab coat
- Safety glasses
- 70% ethanol spray
- Double-stranded DNA of any accessible form (Lyophilized pellets or flakes of DNA preferred) ~ 5g
- Nuclease-free water
- Sodium Acetate (NaOAc) ~ 500g
- Glycogen (20mg/mL stock) ~ 100µL per X stock
Troubleshooting
Methodology
This derivation applies the Beer-Lambert Law to convert spectrophotometric absorbance (A) into the molarity of double-stranded human somatic DNA. The molar output served as the input for subsequent electrostatic and dielectric modelling of ethanol-mediated precipitation.
Beer-Lambert substitution
As per the Beer-Lambert Law (Swinehart, 1962), let 𝑎 = ϵ𝑐𝑙
a: Absorbance (unitless) a = Independent Variable
ϵ: Molar absorptivity ϵ = 50 𝑚𝐿/µ𝑔 𝑐𝑚 (for dsDNA)
c: Concentration of DNA (g/L) c = Dependent Variable
L: Length of light path (cm) L = 0.05 𝑐𝑚
- For all Human Genome DNA, the value for ϵ is 50 𝑚𝐿/µ𝑔 𝑐𝑚. This function applies to any double-stranded DNA (dsDNA) in the human genome.
Rearranging the law,
Substituting values,
Volumetric conversion
Let 𝑚 = 𝑐V
m: mass of solute
c: mass concentration of solution
Vw: Volume of water (assuming negligible volume of dsDNA)
Thus,
Vw = 𝑚/𝑐
Utilizing the value 𝑐 from Beer-Lambert substitution,
Recall that the mass in the numerator will cancel units with the grams in the denominator.
Electrostatic interaction - Coulomb’s law
The volume of the DNA solution is now defined as a function of absorbance and mass. This model can be extended to encompass electrostatic interactions that govern the precipitation of DNA by ethanol. The dielectric constant, characterized by Columb’s law (Jackson, 1999), is used to quantify the effect of our solvent on the strength of interactions between ions.
Electrostatic interaction - Precipitation onset
The threshold dielectric constant of a water-ethanol mixture before precipitation can be estimated through an approximation based on the volume fractions of ethanol and water. Based on experimental data, this occurs when ethanol constitutes more than 64% of the solution volume.
By forming a weighted average, ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = ϕ𝑒 ϵ𝑒 +ϕ𝑤 ϵ𝑤
ϕ𝑒 : Volume fraction of ethanol ϕ𝑒 : 0.64
ϕ𝑤 : Volume fraction of water ϕ𝑤 : 1 - 0.64 = 0.36
ϵ𝑒 : Dielectric constant of ethanol ϵ𝑒 : 24.5 (UW Dalton Research Group)
ϵ𝑤 : Dielectric constant of water ϵ𝑤 : 80.1 (UW Dalton Research Group)
ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = ϕ𝑒 ϵ𝑒 +ϕ𝑤 ϵ𝑤
ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = (0.64)(24.5) + (0.36) (80.1)
ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = 15.68 + 28.836
ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = 44.5
However, this weighting rule is only a rough approximation and is intended as a general guideline. In practice, the dielectric constant ranges between 30 and 40. For the sake of simplicity, this value will not be included in the equation and will instead be left as a constant (ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛).
Electrostatic interaction - Volumetric calculation
The dielectric constant is directly correlated with the solubility of ionic compounds. If it is low, so is the interference with electrostatic forces. Thus, a lower dielectric constant means ions can attract each other and precipitate. The dielectric constants are related to volume by their weighted averages.
ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑉𝑒 * ϵ𝑒 + 𝑉𝑤 * ϵ𝑤 / 𝑉𝑒 + 𝑉𝑤
𝑉𝑒 : Volume of ethanol 𝑉𝑒 = Variable
𝑉𝑤 : Volume of water 𝑉𝑤 = Variable
ϵ 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 : Dielectric constant of the solution ϵ 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = Constant
ϵ𝑒 : Dielectric constant of EtOH ϵ𝑒 = 24.5 (Dalton Research Group)
ϵ𝑤 : Dielectric constant of H2O ϵ𝑤 = 80.1 (Dalton Research Group)
The effective dielectric constant can be determined using a linear, volume-weighted average, targeting a dielectric constant of 40.0.
Utilizing the value for 𝑉𝑤 from Section: Volumetric conversion,
Conclusion and Limitations
Therefore, the volume of ethanol required to precipitate DNA in a solution of water is a function of the dsDNA mass and solution absorbance, as given by the following equation:
Limitations:
- Only works with dsDNA
ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 is left as a constant, as described by Section: Precipitation onset.
The rest of the methodology outlines a two-part approach: first, determining the dielectric constant, and second, validating our theoretical model for ethanol-based DNA precipitation.
Determination of Dielectric Threshold for DNA precipitation
2h 35m
Extract DNA samples from cell cultures
- The samples are more concentrated than our target concentration values, allowing us to dilute the samples with nuclease-free water to the target concentration.
Prepare Water/DNA/Sodium Acetate samples
| A | B | C | D | E | |
| Solution X | |||||
| 1 | 2 | 3 | 4 | ||
| Water (μL) | 100μL | 100μL | 100μL | 100μL | |
| DNA (ng) | 1000ng | 1300ng | 1600ng | 2000ng | |
| Concentration (𝑚DNA/V) | 10ng/μL | 13ng/μL | 16ng/μL | 20ng/μL | |
| Sodium Acetate (mg) | 2.46mg | 2.46mg | 2.46mg | 2.46mg | |
| Glycogen (taken from 20 mg/mL sample) | 100μL | 100μL | 100μL | 100μL | |
Arrange 240 tubes in a test tube rack, each filled with 100 µL of nuclease-free water (60 per concentration).
Dissolve mass of DNA corresponding to the target values listed in Table X in 100 µL of water. Prepare = 20 samples for each concentration, three iterations. All solutions were determined with the spectrophotometer strength in mind.
- The concentration stock given was 33.2 µL for a pure DNA dissolved sample.
Mix all samples of each concentration with various volumes of ethanol around the previously empirically determined concentration of 70% (Sambrook & Russell, 2001).
| A | B | C | D | E | F | G | H | I | J | K | L | M | |
| Solution Y1 | |||||||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
| Solution X (Vx) | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | |
| Ethanol (μL) (95% stock) | 0 | 131.7 | 143.6 | 156.8 | 171.4 | 187.9 | 206.5 | 227.6 | 251.9 | 280 | 313 | 352.4 | |
| Concentration (%EtOH) | 0% | 54% | 56% | 58% | 60% | 62% | 64% | 66% | 68% | 70% | 72% | 74% | |
| Volume (Vy (μL)) | 100 | 231.7 | 243.6 | 256.8 | 271.4 | 287.9 | 306.5 | 327.6 | 351.9 | 380 | 413 | 452.4 | |
| A | B | C | D | E | F | G | H | I | J | |
| Solution Y2 | ||||||||||
| 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | |||
| Solution X (Vx) | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 100μL | 75μL | <- 75μL due to the microcentrifuge size limit | |
| Ethanol (μL) (95% stock) | 400 | 458.8 | 533.3 | 630.8 | 763.6 | 955.6 | 1257.1 | 1350 | ||
| Concentration (%EtOH) | 76% | 78% | 80% | 82% | 84% | 86% | 88% | 90% | ||
| Volume (Vy (μL)) | 500 | 558.8 | 633.3 | 730.8 | 863.6 | 1055.6 | 1357.1 | 1425 | ||
Calculate the dielectric constants with the weighted formula
ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑉𝑒 * ϵ𝑒 + 𝑉𝑤 * ϵ𝑤 / 𝑉𝑒 + 𝑉𝑤
At SATP, ϵ𝑒 = 24.5 (Dalton Research Group) ϵ𝑤= 80.1 (Dalton Research Group)
With this, we obtain the threshold ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 for precipitation.
Incubate at -20 °C for 01:00:00 .
1h
Centrifuge at 16000 x g, 4°C, 00:30:00 .
30m
Pipette the liquid supernatant that remains above the solid precipitated DNA pellet.
Run a Qubit assay to quantify the amount of DNA residual in the supernatant, noting this for later.
Total DNA = Spectrophotometer-measured quantity in final sample + DNA residual found in supernatant.
Prepare 1000 µL of ethanol wash per tube, comprised of 70% ethanol, to dissolve residual salts and contaminants without redissolving the DNA pellet.
Pipette 1 mL of wash into each sample, and invert the tube to rinse gently.
Centrifuge at 16000 x g, 4°C, 00:30:00 .
30m
Remove the ethanol wash, then air-dry the pellet for 00:02:00 -00:05:00 in a tube with the cap loosely ajar to allow airflow until the pellet loses its sheen. Once the shine has worn off, the pellet is ready for resuspension.
5m
Add 100 µL of nuclease-free water to each precipitated sample to dissolve the purified DNA pellet for usage in the spectrophotometer.
Prepare a blank of nuclease-free water for the spectrophotometer.
Measure A260 absorbance values to determine the DNA concentration.
Utilize initial and final DNA concentration to determine the percentage of DNA precipitated. If it is
above 90%, consider it successfully preserved. If no sample meets this criterion, a flaw is determined to
have occurred in the process of the experiment.
Prepare samples as done in Section: Beer-Lambert substitution (without ethanol). The mass of the DNA in each sample should be given in the table.
Measure the A260 values of each sample (blanking for water, glycogen, and sodium acetate).
Using the ϵ𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛, DNA mass and A260 absorbance value, determine, using purely the model, the volume of ethanol required for precipitation.
Add ethanol at the same quantities as in Section: Volumetric conversion.
Incubate at -20 °C for 00:45:00 - 01:00:00 (flexible; generally good for days).
Centrifuge at 16000 x g, 4°C, 00:30:00 .
30m
Assess the presence of precipitated DNA in each sample, determining which ethanol percentage and volume met the previously determined threshold for precipitation. If a band of values is observed, it may be a gradual process. The lowest value of the band will be used for this assessment.
Observe the validity of predictions and thus prove the model to be valid or invalid (threshold values should match with the model, within an error margin of 2% threshold value).