Bio-Rad Gene Pulser MXcell™ Electroporation System - Manual (Page 15)

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Electroporation Protocol
Gene Pulser MXcell Electroporation Guide
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Materials
Needed
n
Actively growing,
freshly passaged
mammalian cells
— For whole
plate protocols:
16 x 10
6 adherent
cells or 4 x 10 6
cells in suspension
— For mini protocols
(6 well sets): 4 x 10 6
adherent cells or
12 x 10
6 cells
in suspension
n
Cell growth medium
n
PBS
n
Gene Pulser MXcell
electroporation system
n
96-well
electroporation plate
n
Gene Pulser
electroporation
buffer or other
buffer suitable for
electroporation
n
Molecule to
electroporate: siRNA,
such as siLentMer
Dicer-substrate
siRNA duplexes,
or DNA
n
24-well tissue
culture plates
n
Turn on the Gene
Pulser MXcell
n
Select “Pre-Set
Protocols”
n
Select appropriate
protocols and edit if
necessary
n
If the cells are adherent, trypsinize the cells and add medium to
inactivate the trypsin; if the cells are in suspension, skip this step
n
Pellet the cells, remove the medium, and resuspend cells in
PBS by gentle pipeting; count the cells
1. For whole plate protocols: transfer 16 x 10 6 adherent
cells or 4 x 10 6 cells in suspension to a new tube, pellet the
cells, and remove PBS by aspiration.
For mini protocols (6 well sets): transfer 4 x 10 6 adherent
cells or 12 x 10 6 cells in suspension to a new tube, pellet the
cells, and remove PBS by aspiration.
2. Resuspend the cells in 16 ml (4 ml for mini protocol) of Gene
Pulser electroporation buffer (this provides 1x10 6 cells/ml of
adherent cells or x10 6 cells/ml of cells in suspension).
. Add the molecule (siRNA, DNA) to the cell suspension.
4. Pipet 10 µl of cell suspension into the appropriate wells
of an electroporation plate.
. Place the lid on the plate and gently rock the plate back
and forth to wet the electrodes.
6. Place the electroporation plate securely into the plate
chamber, close the lid, and press the PULSE button.
7. Transfer all or 100 μl of the cells from each electroporation
well to the wells of a 24-well tissue culture plate containing
00 µl of growth medium.
Note: using this method allows replication of the experiment, providing
two duplicate 24-well tissue culture dishes.
. Incubate cells at 7°C in a humidified CO
2
incubator until
they are ready to be assayed.
Programming
the Instrument
Setting Up the Instrument
Electr
oporation
Pr
otocol