Macherey-Nagel NucleoSpin 740727.10, Руководство пользователя

Страница: 14 / 21

MACHEREY-NAGEL – 08 / 2013, Rev. 01
14
5 NucleoSpin
®
Plasmid EasyPure protocol
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.
> 12,000 x g ,
30 s
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!
+ 150 μL A1
Resuspend
+ 250 μL A2
Mix
RT, 2 min
+ 350 μL A3
Mix
3 Clarification of lysate
Centrifuge for 3 min at full speed (> 12,000 x g).
> 12,000 x g ,
3 min
NucleoSpin
®
Plasmid EasyPure