Chromosomal DNA extraction from Gram-positive bacteria, V2
Anders Kiledal, Julia A Maresca
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.
Before start
Grow culture to high cell density and prepare all solutions.
Steps
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
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)
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
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
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.
