19
www.bioneer.com
BQ-042-101-05
Revision : 2 (2016-07-04)
e. DNA Extraction from Gram-Positive Bacteria for Mini/Midi/Maxi
1.
(Cell collection)
Collect the bacteria
cells (~ 1x10
9
(mini)/ ~ 5x10
9
(midi)/ ~ 1x10
10
(maxi)) by
centrifugation at 6,000 x (>8,000 rpm in a microcentrifuge) for 10 min (or >3,500 rpm for 20 min).
And completely remove the media by pipetting.
2.
(Resuspension of cell pellet: 2-3)
Add 180 μl (mini)/ 1.8 ml (midi)/ 3.6 ml (maxi) of Gram-Positive
Lysis Buffer (not provided) to the collected cell pellet and completely resuspend by vortex mixer or
pipetting.
(Note) Gram-Positive Lysis Buffer can be prepared by using this formulation: 20 mM Tris-HCl (pH8.0),
2 mM sodium EDTA, 1.2% Triton
®
X-100)
3.
Transfer the resuspended cell pellet to each specific tube format.
a. (Mini) Transfer the resuspended pellet to a 1.5 ml or 2 ml tube.
b. (Midi) Transfer the resuspended pellet to a 15 ml tube.
c. (Maxi) Transfer the resuspended pellet to a 50 ml tube.
4.
(Lysis: 4-9)
Add 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of lysozyme (100 mg/ml, not provided) and
mix thoroughly using a vortex mixer.
5.
If RNA-free genomic DNA is required, add up to 10 μl (mini)/ 75 μl (midi)/ 150 μl (maxi) of RNase A
(see “Before you begin”).
6.
Incubate at 37℃ for 30 min.
7.
Add 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of proteinase K (see “Before you begin”).
8.
Add 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) of Buffer ② (Binding) to the tube and mix immediately and
thoroughly using a vortex mixer.
9.
Incubate at 60℃ for 30 min or until the cells are completely lysed.
10.
Go to step 7 of “DNA Extraction from Animal Tissue” in page 14 and continue the extraction process.
