Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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4. 1 M Na 2CO 3.
5. Spectrophotometer.
6. Luciferase assay kit (for example, Luciferase Assay System, Promega).
7. Luminometer, available from many companies including Turner Designs and
PharMingen.
8. Luminometer cuvets.
3. Methods
3.1. Electroporation
The efficiency of transfection is dependent on the growth phase of the cells.
For optimal transfections, grow the cells so that they are in mid-log growth
phase the day of transfection. Keep the cells sterile throughout the following
procedure.
1. Prewarm the cell-culture medium to 37°C, and for each transfection aliquot 10 mL
of prewarmed cell-culture medium to a 25 cm 2 (50 mL) cell-culture flask and place
it in the 37°C carbon dioxide incubator.
2. For each transfection, aliquot the following DNA solutions in a sterile 1.5-mL tube:
a. 10 µg of expression plasmid containing the cDNA, or 10 µg of “empty”
expression plasmid (without the cDNA) as a control (see Note 3).
b. 2 µg of internal control plasmid for normalization of transfections.
c. 2 µg of luciferase reporter plasmid.
3. Count the cell density of the cell culture. Ten million cells are needed for each
transfection.
4. Centrifuge enough cells for your transfections at 250g for 10 min at room temperature.
Be aware that you might lose cells after centrifugation.
5. Resuspend the cells in prewarmed cell-culture medium (see Note 4) to an
approximate density of 30 million cells/mL. Count the cells again and dilute them
in prewarmed medium to a final density of 20 million cells/mL.
6. In the 1.5-mL tubes from step 2, mix 0.5 mL of cell suspension (i.e., 10 million
cells) with the DNA by gentle pipetting. The cells settle easily, so gently mix the
cell suspension before taking each 0.5-mL aliquot. Transfer the cell/DNA mixture
to a sterile electroporation cuvet.
7. Preincubate the cuvets for 5 min if necessary (see Note 5).
8. Set up the electroporation system (pulse generator) by selecting a voltage suitable
for the cells (see Note 6). Adjust the capacitance or length of time of the
electric pulse according to the manual of the pulse generator. For example, using
the Gene Pulser ® II system (Bio-Rad), set the capacitance to 950 µF, resulting in
an electric pulse time of 15–20 ms.
9. Be sure to remove all liquid from the outside of the cuvet, for example, by drying
with a tissue, before electroporating. Just before inserting the cuvet into the shocking
chamber, gently flick it a couple of times to mix the cells. Electroporate the cells.
10. After electroporation allow the cells to recover by letting them stand at room
temperature for 5 min (see Note 5).