May 01, 2025

Public workspaceEco-Friendly Efficient DNA Staining Using Modified Methylene Blue Solution: A Frugal and Safer Alternative to Ethidium Bromide in Low-Resource Biotechnology Laboratories

DOI
dx.doi.org/10.17504/protocols.io.dm6gp27p8lzp/v1
  • Joseph shenekji1,
  • Kamar Shayah1,
  • Sara Shanta1,
  • Ladi Serawan1,
  • Elaf Bobaky1,
  • Ahmad Mouzayek1
  • 1Department of Biotechnology Engineering, Faculty of Technical Engineering, University of Aleppo
  • Joseph shenekji: PhD in Biotechnology Engineering
  • Kamar Shayah: PhD in Biotechnology Engineering
  • Sara Shanta: Graduate student in Biotechnology Engineering
  • Ladi Serawan: Graduate student in Biotechnology Engineering
  • Elaf Bobaky: Graduate student in Biotechnology Engineering
  • Ahmad Mouzayek: Graduate student in Biotechnology Engineering
Joseph shenekji
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp27p8lzp/v1
Protocol Citation: Joseph shenekji, Kamar Shayah, Sara Shanta, Ladi Serawan, Elaf Bobaky, Ahmad Mouzayek 2025. Eco-Friendly Efficient DNA Staining Using Modified Methylene Blue Solution: A Frugal and Safer Alternative to Ethidium Bromide in Low-Resource Biotechnology Laboratories. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp27p8lzp/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
We use this protocol and it's working
Created: May 01, 2025
Last Modified: May 01, 2025
Protocol Integer ID: 206713
Keywords: DNA, staining, methylene blue, low-cost, eco-friendly
Abstract
This protocol presents a validated, low-cost, and environmentally sustainable method for visualizing DNA using methylene blue staining after agarose gel electrophoresis. It eliminates the need for carcinogenic dyes like ethidium bromide and does not require UV transilluminators, knowing that safe dyes like SYBR safe are considered expensive for low-budget labs, making this method especially suitable for educational and research biotechnology laboratories in developing regions, in Aleppo in our case.

Materials
Materials and Reagents

AB
ReagentPurpose
Methylene blue (powder)Intercalating dye that binds to nucleic acids
Sodium acetate (CH₃COONa)Enhances DNA-dye retention via ionic stabilization and pH control
Glacial acetic acidAdjusts pH for optimal dye-DNA interaction (acidic range)
Sodium chloride (NaCl)Increases ionic strength, promoting stable dye binding to DNA
Ethanol (99%) or Acetic acid 5%Fixation of stained DNA to prevent fading
Distilled water (dH₂O)Solvent and diluent
Loading dyeAdds density and tracking dyes to DNA samples for gel loading
AgaroseMedium for DNA electrophoresis
TAE or TBE bufferElectrophoresis running buffer
DNA ladder/markerReference for molecular weight visualization
Equipment
  • Electrophoresis apparatus with power supply
  • Gel casting tray and combs
  • Light box or white-light transilluminator
  • pH meter or pH paper
  • Balance (precision: ±0.001 g)
  • Pipettes and microcentrifuge tubes
  • Beakers, measuring cylinders
  • Heating source (optional for increasing stain uptake)
Introduction
Introduction
Ethidium bromide (EtBr), though widely used for DNA staining, poses significant health and environmental risks due to its mutagenicity and the need for UV light exposure. Methylene blue offers a non-toxic, UV-free alternative. This protocol describes an optimized approach for using methylene blue to visualize DNA ladder bands, focusing on the molecular roles of each component for improved clarity and reproducibility.

Preparation of Enhanced Methylene Blue Staining Solution
Preparation of Enhanced Methylene Blue Staining Solution
Prepare 0.5 M Sodium Acetate (pH 5.5):
To prepare a robust, low-toxicity staining solution that maximizes DNA interaction.
  • WeighAmount4.1 g sodium acetate and dissolve in ~Amount80 mL distilled water.
  • Adjust pH toPh5.5 using glacial acetic acid (kitchen vinegar could do just fine).
  • Bring volume to Amount100 mL .
Molecular Role: Sodium acetate maintains an acidic environment that enhances the electrostatic binding between methylene blue and DNA phosphate backbones.
0.25 gr methylene bluePreparation of Methylene Blue Stock Solution:
  • Weigh Amount0.25 g Amount0.25 g methylene blue and dissolve in Amount1 L Amount1 L distilled water.
0.25 gr methylene blue
dissolving methylene blue in 1 L of distilled water
  • Add Amount100 mL Amount100 mL ofConcentration0.5 Molarity (M) Concentration0.5 Molarity (M) sodium acetate (final concentration ~Concentration0.05 Molarity (M) Concentration0.05 Molarity (M) ).
  • Optional: Add Concentration0.1 Molarity (M) Concentration0.1 Molarity (M) NaCl (0.58Amount0.58 g Amount0.58 g g/100 mL) to stabilize staining.
Molecular Role: Methylene blue intercalates into the minor groove of DNA, especially under acidic conditions where the positive charge on the dye is stabilized.

Optional Fixative Preparation:
  • 10% Ethanol: Mix Amount10 mL Amount10 mL ethanol + Amount90 mL Amount90 mL water.
  • 5% Acetic Acid: MixAmount5 mL Amount5 mL glacial acetic acid + Amount95 mL Amount95 mL water.
DNA Electrophoresis
DNA Electrophoresis
1h 57m
1h 57m
Prepare 1% Agarose Gel:
  • Dissolve Amount1 g agarose in Amount100 mL TAE buffer.
  • Heat until fully melted.
  • Cool slightly, then pour into casting tray with comb.
loading ladder markerLoad DNA Ladder:
  • Mix 5 µL loading dye with DNA ladder. (1kb Gene Ruler Fermentas)
  • Load samples into wells and run gel at:
  • 50 V forDuration00:05:00 , then
  • 100 V for Duration00:30:00 .
loading ladder marker
Function: Electrophoresis separates DNA based on molecular weight.

5m
  • Staining the Gel
  • Post-Electrophoresis Staining:
  • Immerse gel in prepared methylene blue staining solution.
  • Stain at room temperature for Duration00:20:00 , or for Duration01:00:00 for enhanced sensitivity in lower temperatures in the winter.
staining solution
Optional Fixation (Recommended):
Soak stained gel in Concentration10 % (v/v) ethanol orConcentration5 % (v/v) acetic acid forDuration00:10:00 to fix bands, you could put Amount10 mL of ethanol or Amount5 mL acetic acid to the same solution of staining containing methylene blue.
1h 30m
Destaining:
  • Rinse gel in dH₂O or 1X TAE for Duration00:15:00 .
  • Change wash solution every Duration00:05:00 until background is sufficiently clear.
  • use Temperature30 °C c water to have faster results.
20m
Visualization and Interpretation
  • Visualize DNA bands under white light or a light box. (we used a white plexiglass and a fixed a light under it).
  • Bands appear as dark blue lines against a lighter background.
  • Faint bands may require extended destaining or longer staining time.
  • in case you rinsed too much and the bands disappeared, you can simply put the gel back into staining solution for 5-10 minutes and rinse for Duration00:02:00 , and the bands will reappear.
  • you could use smartphone image editing apps to enhance contrast.

2m
Troubleshooting and Optimization

ABC
IssueCauseSolution
Faint bandsInsufficient staining time or low DNA amountIncrease staining time or DNA concentration
High backgroundIncomplete rinsing or too high dye concentrationIncrease washing steps, optimize dye concentration
Bands fade quicklyNo fixation step appliedUse 10% ethanol or 5% acetic acid to fix dye
Uneven stainingPoor mixing or uneven gel immersionEnsure uniform staining solution coverage
Troubleshooting several problems

Comparison Table: Methylene Blue vs. Ethidium Bromide

ABC
FeatureMethylene BlueEthidium Bromide
CostLowHigh
SafetyNon-toxicCarcinogenic
UV Light RequiredNo (white light suffices)Yes
Environmental ImpactEco-friendlyHazardous waste
Staining Time20 - 30 minutes15–30 min
Band PersistenceLong-lasting (if fixed)Fades quickly under UV
SensitivityModerateHigh
Ease of DisposalSimpleRequires hazardous disposal
comparison between EtBr and Methylene Blue

Results
Results
Interpretation of Gel Electrophoresis Results Using Ethidium Bromide and Methylene Blue Compared to a Commercial DNA Ladder
comparing DNA staining using EtBr, Methylene blue, and Commercial standard.
This image presents a comparative analysis of DNA band visualization using two common staining methods-ethidium bromide (EtBr) and methylene blue-on a 1% agarose gel in TAE buffer, alongside a commercial 1 kb DNA ladder (GeneRuler, Fermentas) as a molecular weight reference.

Band Patterns and Sensitivity

The EtBr-stained lane displays 14 distinct bands, visualized as bright orange-yellow under UV illumination. Ethidium bromide intercalates between DNA bases, providing high sensitivity and clear band definition, especially for fragments of medium and lower molecular weight.

The methylene blue-stained lane reveals 12 visible bands, appearing as dark blue bands on a lighter blue background. Methylene blue enables DNA visualization under normal white light, offering a safer alternative to EtBr, though with slightly reduced sensitivity, particularly for smaller DNA fragments.

The reference DNA ladder lane contains 15 bands, corresponding to fragment sizes ranging from 75 to 20,000 base pairs, with key marker bands (5000, 1500, 500 bp) highlighted for orientation. Red arrows in the image indicate the correspondence between visible bands in the methylene blue lane and their respective sizes in the ladder.

Comparative Analysis
Ethidium bromide demonstrates higher sensitivity, allowing detection of more bands and lower DNA quantities compared to methylene blue. This is evident from the 14 bands visualized with EtBr versus 12 with methylene blue, while the original ladder contains 15 bands.

Methylene blue, while less sensitive, still provides adequate resolution for most DNA fragments, and its bands align accurately with the expected sizes in the commercial ladder, as indicated by the red arrows.

Both staining methods allow for reliable estimation of DNA fragment sizes when compared to the reference ladder, but EtBr is superior for detecting low-abundance or small fragments.

Practical Implications
Ethidium bromide is recommended when maximum sensitivity is required, such as for detecting low-concentration DNA samples. However, due to its mutagenic properties, appropriate safety measures must be implemented.

Methylene blue offers a safer, non-mutagenic alternative suitable for routine applications and for downstream recovery of DNA, albeit with lower sensitivity for small or faint bands.
The choice of stain should be guided by experimental requirements: EtBr for maximal sensitivity, methylene blue for user safety and DNA integrity.
Conclusion
The comparison demonstrates that while both stains are effective for DNA visualization in agarose gels, ethidium bromide provides greater sensitivity and band resolution, closely approaching the full band pattern of the original commercial ladder. Methylene blue, though less sensitive, remains a practical and safer option for many standard Biotechnology laboratory applications, with low-cost and high availability, it is the best candidate for students labs, in cases where safe DNA stains are not available or expensive.
Protocol references
- Hu, Z., & Tong, C. (2007). Synchronous fluorescence determination of DNA based on the interaction between methylene blue and DNA.. Analytica chimica acta, 587 2, 187-93 . https://doi.org/10.1016/J.ACA.2007.01.050.
- Vardevanyan PO, Antonyan AP, Parsadanyan MA, Shahinyan MA, Hambardzumyan LA. Mechanisms for binding between methylene blue and DNA. Journal of Applied Spectroscopy. 2013 Sep;80:595-9.
- Merdekawati F, Gustira Rahayu I, Khoirul Abror Y. Check Similarity: METHYLENE BLUE AS ALTERNATIVE DNA STAINING IN ELECTROPHORESIS. InProceeding of The 5th International Conference on Interprofessional Health Collaboration and Community Empowerment 2022 (Vol. 4, No. 1, pp. 87-92). PPM Poltekkes Kemenkes Bandung M Poltekkes Kemenkes Bandung.