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BQ-042-101-05
Revision : 2 (2016-07-04)
b. DNA Extraction from Cultured Cell for Micro/Mini/Midi/Maxi
1.
Centrifuge the cultured cells (~ 1x10
4
(micro)/ ~ 1x10
6
(mini)/ ~ 5x10
6
(midi)/ ~ 1x10
7
(maxi)) for 10
min at 300 x g. Discard supernatant carefully.
(Note) For cultured cells, “micro” scale is included to accommodate the use of lower cell number
than 1x10
4
cells. Thus, given volume amount of this kit component is adjusted and mentioned on this
procedure.
2.
Resuspend the pellet in 100 μl (micro)/ 200 μl (mini)/ 1 ml (midi, maxi) of 1X PBS
and transfer them to
each specific tube format.
a. (Micro/Mini) Transfer the resuspended pellet to a 1.5 ml or 2 ml tube.
b. (Midi/Maxi) Transfer the resuspended pellet to a 15 ml tube.
3.
Add 10 μl (micro)/ 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of proteinase K (see “Before you begin”) to
the tube.
4.
If RNA-free genomic DNA is required, add up to 2 μl (micro)/ 10 μl (mini)/ 75 μl (midi), 150 μl (maxi) of
RNase A (see “Before you begin”) and incubate for 5 min at room temperature.
5.
(Lysis: 5-6)
Add 100 μl (micro)/ 200 μl (mini)/ 1 ml (midi, maxi) of Buffer ② (Binding) to the sample
and mix immediately and thoroughly using a vortex mixer. You must completely resuspend the sample
to achieve maximum lysis efficiency.
6.
Incubate at 60℃ for 10min.
7.
(DNA precipitation)
Add 200 μl (micro)/ 400 μl (mini)/ 2 ml (midi, maxi) of absolute ethanol and mix
well by vortex mixer or pipetting.
8.
(DNA binding with Magnetic Nano Bead: 8-10)
Add 100 μl (micro, mini)/ 500 μl (midi)/ 1 ml (maxi) of
Magnetic Nano Bead solution
to the tube and mix thoroughly using a vortex mixer until the beads are
fully resuspended.
(Note) Magnetic Nano Bead
Solution contains magnetic nano beads. Please shake well before use.
