Dec 15, 2025

Kit-Free bacterial gDNA extraction V.2

This  protocol  is a draft, published without a DOI.
Kit-Free bacterial gDNA extraction
  • 1University of Oslo
Andreas Sagen
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Protocol Citation: Andreas Sagen 2025. Kit-Free bacterial gDNA extraction. protocols.io https://dx.doi.org/Version created by Andreas Sagen
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: December 24, 2024
Last Modified: December 15, 2025
Protocol  Integer ID: 117101
Keywords: DNA, gDNA, kit-free, salt-out, precipitation, free bacterial gdna extraction, genomic dna from bacteria, dna from salt, genomic dna, molecular weight genomic dna, dna, dna precipitation, bacteria, free of impurity
Abstract
This is an easy and cheap protocol to extract genomic DNA from bacteria based on salt-out to remove proteins and DNA precipitation to isolate DNA from salts. Using this protocol, you should be able to extract high-molecular weight genomic DNA, free of impurities and at a high concentration.
Materials
Centrifuge
Heat block
Freezer
Before start
Preheat the heat block to 37 °C. Get some ice, and thaw reagents and enzymes.
Lysis
1h 25m
Pellet a 10 mL overnight bacterial culture by centrifugation with 6 000 rcf for 10 minutes at 4°C and decant supernatant as waste
10m
Resuspend pellet in 1 000 µL ice-cold PBS (w/o Ca2+, Mg2+) and transfer suspension to a 1.5 mL reaction tube
Pellet the suspension by centrifugation with 10 000 rcf for 5 minutes at room temperature and decant supernatant as waste
5m
Step case

Gram-negative
15 steps

Due to the structural differences in gram-negative and gram-positive bacterial cell walls, it is necessary to treat these bacteria different for optimal lysis. The gram-negative cell wall is easier to lyse, and as such less lysozyme and surfactant is necessary to free the genomic DNA from the cells.
Resuspend pellet in 346 µL PBS (w/o Ca2+, Mg2+), 20 µL 20 mg/mL lysozyme, 10 µL 0.5 M EDTA (pH 8.0) and 4 µL 10 mg/mL RNase A
Incubate the suspension for 30 minutes at 37°C with 250 rpm shaking
30m
Add 50 µL 10% SDS, 10 µL 20 mg/mL proteinase K and 40 µL 5 M NaCl. Mix solution by tube inversion
Incubate suspension for 30 minutes at 56°C with 2 000 rpm shaking

Note
Incubation for 30 minutes is usually enough, but if lysate is not clear (or near clear) after 30 minutes incubation, extend the incubation for several hours.

30m
Add 360 µL 5 M NaCl and 40 µL 4 M KCl. Mix solution by tube inversion
Incubate solution on ice for 5 minutes
5m
Centrifuge solution at 14 000 rcf for 5 minutes at room temperature. Decant supernatant into a 5 mL reaction tube
5m
DNA precipitation
3h 7m
Add 100 µL 3 M sodium acetate (pH 5.2) and 2 500 µL ice-cold absolute ethanol
Incubate solution at -20°C for minimum 2 hours

Note
Incubate overnight to increase yield, especially important if the DNA concentration is low.

2h
Centrifuge solution at 14 000 rcf for 30 minutes at 4°C. Decant supernatant as waste
30m
Resuspend pellet in 1 000 µL ice-cold 70% ethanol. Incubate for 10 minutes at room temperature
10m
Centrifuge solution at 14 000 rcf for 10 minutes at 4°C. Decant supernatant as waste
10m
Centrifuge again at 14 000 rcf for 2 minutes at 4°C. Remove residual ethanol by pipette
2m
Air-dry pellet until all ethanol has evaporated
15m
Resuspend pellet in 200 µL T10E0.1 buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)