Dec 24, 2024
Version 1
Kit-Free bacterial gDNA extraction V.1
This protocol is a draft, published without a DOI.
- 1University of Oslo
Protocol Citation: Andreas Sagen 2024. Kit-Free bacterial gDNA extraction. protocols.io https://protocols.io/view/kit-free-bacterial-gdna-extraction-dm7z49p6
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: September 26, 2024
Last Modified: December 24, 2024
Protocol Integer ID: 108505
Keywords: DNA, gDNA, kit-free, salt-out, precipitation
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
Lysis
3h
3h
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
Gram-negative15 steps
This is a general process to degrade the gram-negative cell wall. Look through the other steps to find genus/species specific protocols.
Resuspend pellet in 336 µL PBS (w/o Ca2+, Mg2+), 20 µL 20 mg/mL lysozyme, 40 µL lysis enhancement solution (200 mM Tris-HCl, 20 mM EDTA, 12% Triton X-100) 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 1400 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.
2h
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
DNA precipitation
4h
4h
Add 100 µL 3 M sodium acetate (pH 5.2) and 2500 µL ice-cold 96% 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
30m
Resuspend pellet in 200 µL T10E0.1 buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)