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5
NucleoSpin
Before starting the preparation:
• Check if Wash Buffer AQ was prepared according to section 3.
1
Cultivate and harvest bacterial cells
Use 2–10 mL of a saturated E. coli culture (see
page 8, table 3), pellet cells in a standard benchtop
microcentrifuge for 30 s at > 12,000 x g.
Discard the supernatant and remove as much of the
liquid as possible.
2
Cell lysis
Add 150 μL Buffer A1. Resuspend the cell pellet
completely by vortexing or pipetting up and down. Make
sure no cell clumps remain before addition of Buffer A2!
Attention: Check Buffer A2 for precipitated SDS. If a white
precipitate is visible, warm the buffer for several minutes
at 30–40 °C until precipitate is dissolved completely. Cool
buffer down to room temperature (18–25 °C) before use.
Add 250 μL Buffer A2. Mix gently by inverting the tube
5 times. Do not vortex to avoid shearing of genomic DNA.
Incubate at room temperature (18–25 °C) for up to 2 min
or until lysate appears clear.
Add 350 μL Buffer A3. Mix thoroughly by inverting the
tube until LyseControl has turned colorless throughout
the lysate without any traces of blue color. Do not vortex
to avoid shearing of genomic DNA!
3
Clarification of lysate
Centrifuge for 3 min at full speed (> 12,000 x g).
14
NucleoSpin
Plasmid EasyPure
®
Plasmid EasyPure protocol
®
MACHEREY-NAGEL – 08 / 2 013, Rev. 01
> 12,000 x g,
30 s
+ 150 μL A1
Resuspend
+ 250 μL A2
Mix
RT, 2 min
+ 350 μL A3
Mix
> 12,000 x g,
3 min
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