Jul 03, 2025

Public workspaceComprehensive Workflow for Extracting High-Quality Bacterial DNA from Milk Samples

DOI
dx.doi.org/10.17504/protocols.io.8epv5k78dv1b/v1
Comprehensive Workflow for Extracting High-Quality Bacterial DNA from Milk Samples
  • Panagiota STATHOPOULOU1,
  • Ioannis Galiatsatos1,
  • Maria Simoni1,
  • Nikolaos D. Andritsos1
  • 1University of Patras
  • Lab of Systems Microbiology and Applied Genomics
Ioannis Galiatsatos
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5k78dv1b/v1
Protocol Citation: Panagiota STATHOPOULOU, Ioannis Galiatsatos, Maria Simoni, Nikolaos D. Andritsos 2025. Comprehensive Workflow for Extracting High-Quality Bacterial DNA from Milk Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5k78dv1b/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: July 03, 2025
Last Modified: July 03, 2025
Protocol Integer ID: 221677
Keywords: milk, dna extraction, automated, manual, dna purification, bacterial dna from milk, quality bacterial dna from milk sample, accurate representation of the milk microbiome, milk microbiome, quality bacterial dna, bacterial dna, automated purification step, milk sample, difficult matrix for dna extraction, bulk of milk, final purification, certain bacterial species, dna, milk fat fraction, bacterial cell, extraction, using cetyltrimethylammonium bromide, protein, purification
Abstract
This protocol provides a comprehensive workflow for extracting high-quality bacterial DNA from milk samples. Milk is a notoriously difficult matrix for DNA extraction due to high concentrations of inhibitors like fats and proteins (casein). Standard kit protocols often fail because they are not designed to break down this complex matrix effectively. This procedure combines two powerful methods: a robust manual pre-treatment using Cetyltrimethylammonium bromide (CTAB) to effectively lyse bacterial cells and remove the bulk of milk's inhibitors, and an automated purification step using the RBC Biosciences MagCore® Bacterial Kit to efficiently bind, wash, and elute the DNA using magnetic-particle technology. To ensure the most accurate representation of the milk microbiome, this protocol includes an advanced option to co-process the milk fat fraction, where certain bacterial species are known to reside. By preparing a clean, inhibitor-free lysate manually, you can then use the MagCore® system for the final purification, ensuring higher yield and purity.
Guidelines
Understanding the Protocol's Purpose This protocol is specifically designed to overcome the challenges of extracting bacterial DNA from milk, a sample type known for high levels of inhibitors like fats and proteins. It combines a robust manual pre-treatment to clean the sample with an automated system for final DNA purification, aiming for higher yield and purity than standard kits alone.
Key Decisions During the Protocol You will need to make a critical choice after the initial centrifugation of the milk:
  • Option A (Standard Protocol): Choose this for routine analysis. You will only process the cell pellet and discard the fat layer. This is a faster approach.
  • Option B (Comprehensive Protocol): Choose this if you need the most complete representation of the milk's microbial community. This method co-processes the cell pellet and the fat fraction, as some bacteria reside in the fat. Note that this option requires an additional reagent, Tween-20, for the washing step.
Critical Steps & Handling Instructions
Failure to perform these steps correctly can lead to poor results or protocol failure.
  1. Initial Pellet Separation: After centrifuging the milk, be careful when removing the top fat layer and the liquid supernatant to avoid disturbing the cell pellet at the bottom.
  2. Casein Dissolution: Do not skip the EDTA incubation step. This is important for breaking down the milk's protein matrix, which can trap bacteria and inhibit downstream reactions. The suspension should become clearer, indicating the step was successful.
  3. Lysis Steps:
  • The Lysozyme step is essential because milk samples often contain Gram-positive bacteria with tough cell walls that need to be broken down.
  • During the CTAB Lysis incubation at 65°C, make sure to invert the tube every 20-30 minutes to ensure thorough mixing and complete cell lysis.
4. Lysate Clarification (Most Critical Step):
  • Before loading your sample into the MagCore
    system, you must centrifuge the lysate at high speed to pellet insoluble debris.
  • Only transfer the clear supernatant to the MagCore
    cartridge. Transferring any of the pelleted debris can interfere with the magnetic beads and cause the automated run to fail.

General Best Practices Sample Volume: Use a larger starting volume of milk (up to 40 mL) if you expect the bacterial count to be low.
Sterility: Use sterile tubes, tips, and reagents (like PBS) to avoid contaminating your sample with outside DNA.
Storage: Once the protocol is complete, store your purified DNA at -20°C or colder for long-term stability.


Materials
Reagents:
- Raw or pasteurized milk
- CTAB Lysis Buffer (see preparation below)
- Phosphate-Buffered Saline (PBS) or Tris-EDTA (TE) Buffer (pH 8.0)
- 50-100 mM EDTA solution (pH 8.0)
- Tween-20 (for comprehensive protocol option)
- Nuclease-free water
- Phenol
-Chloroform
-Isopropanol

From RBC MagCore® Bacterial DNA Kit (Cat.No. MBB-01/MBB-02):
- Pre-filled reagent cartridges
- Lysozyme and Lysozyme Reaction Buffer
- Proteinase K and PK Storage Buffer

Equipment:
- High-speed refrigerated centrifuge
- 50 mL and 1.5/2.0 mL microcentrifuge tubes
- Water bath or heating block set to 37°C and 65°C
- Vortex mixer
- Micropipettes and sterile tips
- Sterile swabs or spatulas
- RBC Biosciences MagCore® Instrument
Troubleshooting
Safety warnings
Phenol and chloroform are highly toxic and corrosive. Perform this step in a fume hood with appropriate personal protective equipment (gloves, lab coat, eye protection).
Before start
  1. Reagent Preparation:
  • The CTAB Lysis Buffer must be prepared according to the recipe. Crucially, warm it to 65°C before use to ensure all components are fully dissolved.
  • The Lysozyme solution must be prepared fresh right before you need it for the lysis step.
2. Equipment Setup:
  • Ensure your refrigerated centrifuge can reach the required speeds (e.g., 10,000 x g).
  • Have two water baths or heating blocks ready, one set to 37°C (for lysozyme) and another to 65°C (for CTAB lysis).

Buffer Preparation
1h
CTAB Lysis Buffer (2% w/v):
1h
To prepare Amount100 mL , dissolve the following in nuclease-free water: Amount2 g CTAB (Cetyltrimethylammonium bromide), Amount8.18 g NaCl (for Concentration1.4 Molarity (M) final concentration), Amount10 mL of 1 M Tris-HCl (for 100 mM final concentration, Ph8 ), Amount4 mL of Concentration0.5 Molarity (M) EDTA (for Concentration20 millimolar (mM) final concentration, Ph8 ).
Adjust the final volume to Amount100 mL with nuclease-free water.

Warm to Temperature65 °C before use to ensure the CTAB is fully dissolved.

Part 1: Manual Pre-Treatment – Sample & Inhibitor Removal
57m
Sample Collection & Centrifugation:
10m
Centrifigation
Pour Amount10-40 mL of milk into a Amount50 mL centrifuge tube. A larger starting volume is recommended for low-biomass samples.

Centrifuge at Centrifigation10.000 x g, 4°C, 00:15:00 to pellet bacterial and somatic cells.

15m
Separating Milk Fractions:
After centrifugation, three layers will be visible: a solid fat layer on top, the liquid skim milk, and a cell pellet at the bottom.
Choose one of the following two paths:
2m
Critical
Option A (Standard Protocol - Pellet Only): For routine analysis, carefully remove and discard the top fat layer and the middle liquid supernatant, leaving only the cell pellet. Proceed to Step 4.
Option B (Comprehensive Protocol - Pellet + Fat Fraction): For the most accurate representation of the entire milk microbiome, it is important to process the fat fraction, as some bacterial species preferentially associate with it. Use a sterile spatula to carefully transfer the top fat layer into a new, clean 50 mL centrifuge tube. Aspirate and discard the middle liquid supernatant from the original tube, leaving the cell pellet. Resuspend the cell pellet in Amount1-2 mL of sterile PBS and transfer this suspension into the tube containing the fat fraction. This combines the two key fractions for co-extraction. Proceed to Step 4.

Pellet Washing & Detergent Wash:
5m
If you chose Option A: Resuspend the pellet in Amount5-10 mL of sterile PBS.

If you chose Option B: Resuspend the combined fat and pellet mixture in Amount5-10 mL of sterile PBS containing 0.1% Tween-20 to help dissolve residual fat and release associated bacteria. Vortex thoroughly. Centrifuge at Centrifigation10.000 x g, 00:10:00 . Discard the supernatant. Perform one additional wash on the pellet using plain PBS to remove residual detergent. Centrifuge and discard the supernatant.

10m
Centrifigation
Casein Dissolution (Recommended):
Incubation
Optional
To break down the protein matrix and release trapped bacteria, resuspend the washed pellet in Amount1 mL of Concentration50-100 millimolar (mM) EDTA solution (Ph8 ).

Incubate at room temperature for Duration00:10:00 . The milky suspension should become clearer as casein micelles dissolve.

10m
Incubation
Centrifuge at >Centrifigation12.000 x g, 00:05:00 to pellet the cells. Carefully discard the supernatant.

5m
Centrifigation
Part 2: Manual Pre-Treatment – Lysis
2h 40m
Gram-Positive Lysis (Lysozyme): This step is essential for milk samples, which are likely to contain Gram-positive bacteria with tough cell walls.
Prepare Lysozyme Solution: Freshly prepare a Concentration20 mg/mL Lysozyme solution by dissolving Lysozyme powder in the Lysozyme Reaction Buffer provided with the kit.

Resuspend Pellet: Resuspend the cell pellet in Amount200 µL of the freshly prepared Lysozyme solution. Incubate at Temperature37 °C for Duration01:00:00 , mixing occasionally.

1h
RNA Removal (RNase Treatment)
After the lysozyme incubation, add Amount4 µL of RNase A to the sample mixture.

Vortex briefly to mix.
Incubate at room temperature for 1Duration00:10:00 .

10m
Incubation
CTAB Lysis & Protein Digestion:
To the lysozyme-treated sample, add Amount500 µL of pre-warmed (Temperature65 °C ) CTAB Lysis Buffer.

Add Amount20 µL of reconstituted Proteinase K from the RBC MagCore
kit.

Vortex briefly and incubate at 65°C for 1-2 hours, inverting the tube every 20-30 minutes to mix. The solution will become viscous as cells lyse.
1h 30m
Incubation
Part 3A: Automated Purification – MagCore® Integration
50m
Lysate Clarification:
Critical
After the 65°C incubation, centrifuge the lysate at >Centrifigation13.000 x g, 00:05:00 at room temperature. This will pellet any insoluble debris that could interfere with the magnetic beads. The clear liquid supernatant contains your DNA.

5m
Centrifigation
Load Sample into MagCore® Cartridge:
This is the critical 'hand-off' step. You will bypass the lysis step of the automated protocol. Carefully pipette the clear supernatant from Step 9 into the Lysis Buffer well (Well 11) of the RBC MagCore® pre-filled cartridge. Do not transfer any of the pelleted debris.
Add Magnetic Beads:
Using a clean pipette tip, transfer the magnetic beads from the 'Beads Mixture' well (Well 9) of the cartridge into the same well containing your lysate (Well 11).
Run Automated Protocol:
Place the prepared cartridge into the MagCore® instrument. Select and run the standard pre-programmed Bacterial DNA extraction protocol (502). The instrument will now automatically perform the DNA binding, washing, and elution steps on your pre-cleaned lysate.
45m
Collect Purified DNA:
Once the run is complete, retrieve the elution tube containing your purified bacterial DNA. Store the DNA at Temperature-20 °C or below for long-term stability.

Part 3B: Purification with Phenol:Chloroform (if the automated solution isn't available)
1h 19m
SAFETY NOTE: Phenol and chloroform are highly toxic and corrosive. Perform this step in a fume hood with appropriate personal protective equipment (gloves, lab coat, eye protection).
Toxic
Cool the lysate to room temperature.
Add an equal volume (approx. Amount700 µL ) of phenol:chloroform:isoamyl alcohol (25:24:1).

Vortex for 10 seconds to create an emulsion, then centrifuge at Centrifigation14.000 x g, 00:10:00 to separate the phases.

10m
Centrifigation
Carefully transfer the upper, clear aqueous phase (which contains the DNA) to a new sterile microcentrifuge tube. Do not disturb the white protein layer at the interface.
Repeat the phenol:chloroform extraction on the aqueous phase until the interface is clean and white, indicating that most proteins have been removed.
DNA Precipitation:
To the final aqueous phase, add 0.7 volumes of ice-cold isopropanol (e.g., if you have Amount500 µL of aqueous phase, add Amount350 µL of isopropanol).

Mix gently by inverting the tube several times. You should see white, stringy DNA precipitate out of the solution.
Incubate at Temperature-20 °C for at least Duration00:30:00 to maximize DNA precipitation. Optionally incubate overnight.

30m
Incubation
Overnight
Pellet DNA:
Centrifuge at Centrifigation14.000 x g, 4°C, 00:15:00 . A small white pellet of DNA should be visible at the bottom of the tube.

15m
Centrifigation
Carefully decant the supernatant without disturbing the pellet.
Wash DNA Pellet:
Add Amount500 µL of ice-cold 70% ethanol to the tube. This step washes away residual salts and CTAB.

Centrifuge at Centrifigation14.000 x g, 4°C, 00:06:00

6m
Centrifigation
Carefully decant the ethanol. Repeat the 70% ethanol wash one more time for higher purity.
Dry and Resuspend DNA:
After the final wash, remove as much ethanol as possible with a pipette.
Air-dry the pellet for 5–10 minutes at room temperature. Do not over-dry the pellet, as this can make it difficult to dissolve.
8m
Resuspend the DNA pellet in 30–50 µL of TE buffer or nuclease-free water. You may need to warm the tube to 65°C for 10 minutes to help dissolve the DNA completely. Store the purified DNA at -20°C.
10m
Protocol references
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  2. Evaluation of milk sample fractions for characterization of milk microbiota from healthy and clinical mastitis cows | PLOS One, https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193671
  3. DNA isolation procedure from cream, dried skim milk and sediment of 200... - ResearchGate, https://www.researchgate.net/figure/DNA-isolation-procedure-from-cream-dried-skim-milk-and-sediment-of-200-ml-raw-milk_fig1_226978572
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  5. Preparation of milk samples for PCR analysis using a rapid filtration ..., https://in.ncu.edu.tw/~jyewu/CV/Download/JAM-2004.pdf
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