Apr 10, 2023
Isolation of bacterial DNA with Gentra Puregene kit (modified protocol)
- 1University of Toronto
Protocol Citation: o.pogoutse, Megan Frederickson 2023. Isolation of bacterial DNA with Gentra Puregene kit (modified protocol). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorwo7v4o/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: March 02, 2023
Last Modified: April 10, 2023
Protocol Integer ID: 77988
Abstract
The Qiagen protocol for purification of genomic DNA from gram-positive bacterial cultures using Yeast/Bact. Kit ( Gentra Puregene Handbook - QIAGEN) was modified to aid in the isolation of bacterial DNA from heterogeneous, low volume, low OD cultures
Cell lysis
Cell lysis
transfer culture to 1.5ml microfuge tube
centrifuge for 10min at 12000rpm to pellet cells
decant or aspirate off supernatant
add 1 ml sterile PBS to pellet and vortex briefly to resuspend cells
centrifuge for 10 min at 12000rpm
decant of supernatant
repeat steps 4-6 one more time
resuspend pellet in 50ul PBS buffer
add 2ul of MetaPolyzyme solution (5mg/ml) (Sigma#MAC4L)
incubate overnight at 35C
add 310ul lysis solution (Qiagen #158113) to each tube. Pipette up and down gently to
resuspend cells
heat samples to 80ºC for 5-10 minutes to complete the lysis
**stop step: lysed cells are stable at room temperature
RNase treatment
RNase treatment
add 1.2ul of 10mg/ml RNase A per tube
invert tube gently to mix
incubate at 37ºC for 30-60 minutes
Protein precipitation
Protein precipitation
cool sample to room temperature
add 105ul of protein precipitation solution (Qiagen #158123)
invert tube gently several times to mix
place on ice for 30 minutes
centrifuge at 12000rpm for 3 minutes.
(The precipitated protein should form a tight pellet).
transfer supernatant to a fresh sterile tube (by pipette)
DNA precipitation
DNA precipitation
add 1-2ul of glycogen (20mg/ml) (Thermofisher #R0561) to the supernatant and mix it by pipetting up and down
add 400ul of 100% isopropanol
invert tube gently 50 times to mix
(optionally: incubate at RT for 30 min to improve DNA yield)
pellet at 13000rpm for 3 minutes
carefully decant off isopropanol (the pellet might be loose)
add 500ul of 70% ethanol (cold), and invert tube a few times to wash the DNA pellet
centrifuge at 13000rpm for 3 minutes
pour off ethanol carefully – pellet may be loose
air dry pellet for 15-30 minutes
rehydrate DNA in ~35-50ul of 10mM Tris buffer (pH7.5-8.0)
(adjust for large or smaller pellet)