Miniprep
– 5-PRIME kit
A
method
to
isolate
plasmids
from
transformed
cells during cloning
Procedure
1. Pellet 1.5
ml of fresh
bacterial culture at maximum speed for 1 minute in the provided 2
ml
Culture
Tube.
2.
Remove medium by decanting, taking care not to disturb bacterial pellet.
3. Add
400
µl of
ICE-COLD Complete Lysis Solution.
4. Mix
thoroughly by constant vortexing at the highest setting for a full 30
seconds. This step is
critical for
obtaining maximum yield.
5.
Incubate the lysate at room temperature for 3 minutes.
6.
Transfer the lysate to a Spin Column Assembly by decanting or pipetting.
7.
Centrifuge the Spin Column Assembly for 60 seconds at maximum speed.
8.
Add
400
µl of DILUTED Wash Buffer to the
Spin Column Assembly.
9.
Centrifuge the Spin Column Assembly for 60 seconds at maximum speed.
10.
Remove the Spin Column from the centrifuge and decant the filtrate from
the
Spin Column Assembly Waste Tube. Place the Spin Column back into the
Waste Tube
and return it to the centrifuge.
11.
Centrifuge at maximum speed for 1 minute to dry the Spin Column.
12.
Transfer the Spin Column into a Collection Tube.
13.
Add 50
µl of Elution Buffer directly to
the center of the Spin Column membrane and cap the Collection Tube over
the
Spin Column.
14.
Centrifuge at maximum speed for 60 seconds.
15.
Remove and discard the Spin Column.
16.
The eluted DNA can be used immediately for downstream applications or
stored at
-20°C.
Materials required
Overnight
bacterial cultures of successful clones
5-PRIME
(Eppendorf)
Miniprep
kit
200
and 1000
µl pipette tips
1.5 ml microcentrifuge tubes
Equipment required
200
µl micropipetter
1000
µl micropipetter
Benchtop
centrifuge capable of 20,800 x g
Benchtop
vortexer