Jul 10, 2023
Version 3
Chromosomal DNA extraction from Gram-positive bacteria, V3 V.3
- 1University of Delaware, Department of Biological Sciences;
- 2University of Delaware, Department of Civil and Environmental Engineering
Protocol Citation: Anders Kiledal, Julia A Maresca 2023. Chromosomal DNA extraction from Gram-positive bacteria, V3. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl85119l2w/v3Version created by Julia A Maresca
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 06, 2023
Last Modified: July 10, 2023
Protocol Integer ID: 84620
Keywords: Gram-positive, chromosomal DNA, Firmicutes, Actinobacteria, sequencing
Abstract
Extraction of high-molecular-weight DNA from Gram-positive bacterial species, with optional steps for removing surfactants. This DNA is suitable for sequencing and the protocol can be scaled up at least 5-fold. Modified from a protocol by Tina Wecke, LMU-Munich.
Image Attribution
Julia Maresca, University of Delaware
Guidelines
Recommend wearing gloves throughout and working in a biosafety cabinet if possible to prevent contamination. This protocol can be scaled up at least 5-fold. If the isopropanol precipitation step is used, subsequent steps do not have to be scaled up unless the culture volume is substantially larger.
Materials
SOLUTIONS
TEN
- 10 mM Tris-HCl, pH 8.0
- 10 mM EDTA
- 150 mM NaCl
TEN*
- 10 mM Tris-HCL, pH 8.0
- 1 mM EDTA
- 50 mM NaCl
RNAse A
- 20 mg/mL in water
Lysozyme
- 20 mg/mL in water
SDS
- 10% (w/v) in water
Other reagents:
Isopropanol, ethanol (100% and 70%), phenol, chloroform:isoamyl alcohol (24:1), sterile water.
Enzyme solutions should be stored at -20 between uses or prepared freshly. Other solutions can be stored at room temperature.
CONSUMABLES
- microcentrifuge tubes (or larger centrifuge tubes, depending on volume)
- pipetment (P1000, P200, P20)
- pipet tips (P1000, P200, P20)
- Glass Pasteur pipet with tip bent
Safety warnings
Phenol and chloroform:isoamyl alcohol should be handled in a fume hood and the liquid waste and contaminated tubes should be disposed of in accordance with the institution's rules for handling organic solvent waste.
Before start
Grow culture to high cell density and prepare all solutions.
Grow culture
Grow culture
Inoculate 10 mL rich medium from a fresh overnight culture, and incubate at appropriate temperature on shaker. At OD600 of ~0.8-1.0, harvest cells by centrifugation (10 min., 5000 rpm).
Cell lysis
Cell lysis
Resuspend cell pellet in 2 mL TEN (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl).
Add 100 μL lysozyme (20 mg/mL) and incubate for 20 min at 37ºC.
Add 20 μL RNAse (10 mg/mL) and incubate for 3 min at 65ºC.
Add 40 µl SDS, a small scoop of proteinase K and 550 µl TEN*. Vortex, then incubate at 60°C for 2 hours.
Remove surfactants (optional)
Remove surfactants (optional)
IF THE STRAIN PRODUCES A SURFACTANT THAT INTERFERES WITH THE PHASE SEPARATION, Add 0.1 volume 3 M sodium acetate and 1 volume cold isopropanol, mix, and incubate on ice for 20 min. Centrifuge for 10 min at 5000 rpm and decant the supernatant. Then resuspend in 400 μL TEN and 550 μL TEN* and transfer to a microcentrifuge tube.
Phenol & chloroform:isoamyl alcohol extractions
Phenol & chloroform:isoamyl alcohol extractions
Add 900 μL phenol, mix by inversion. Centrifuge for 5 min. at 13000 rpm and transfer the upper phase to a clean microcentrifuge tube.
Re-extract once with phenol (1 volume) and twice with chloroform: isoamyl alcohol (24:1 v/v, 1 volume)
DNA precipitation
DNA precipitation
Transfer upper phase to 10 mL cold 100% ethanol.
Collect DNA by coiling on the end of a glass Pasteur pipet.
Air dry, then resuspend DNA in 100 μL sterile water overnight at 4ºC.