Biomedical Engineering Reference
In-Depth Information
2. One day before transfection, plate HeLa cells in a 10-cm dish
at ~30 % confl uency in medium without antibiotics.
3. On the day of transfection, check to make sure the cells are
healthy and viable at ~50-60 % confl uency.
4. Prepare the transfection mixture by diluting 800 pmol siRNA
(20
l of Opti-MEM
®
I Reduced
Serum Medium. In a separate tube, dilute 16
ʼ
l from 40
ʼ
M stock) in 625
ʼ
ʼ
l of
l of Opti-MEM
®
I Reduced Serum
Medium. After incubating the two tubes at room temperature
for 5 min, mix the two tubes and incubate the mixture at room
temperature for another 20 min to allow formation of siRNA-
Oligofectamine complex.
5. Slowly add the siRNA-Oligofectamine complex dropwise to
cells using a 200-
Oligofectamine in 44
ʼ
l micropipette tip, and incubate the trans-
fected cells in a 37 °C incubator for 22 h.
6. Place sterile coverslips into each well of a six-well dish. 22 h
after siRNA transfection, trypsinize the transfected cells in the
10-cm dish and seed them into each well of the six-well plate
containing sterile coverslips, to achieve a confl uency of ~40 %.
7. Return the plate to the incubator and allow cells to adhere to
the coverslips for at least 3 h before transfection of rescue
constructs.
8. Set up transfection reactions in sterile polystyrene tubes:
(a) Calculate the volume of DNA to be added in each reac-
tion. The amount of DNA needed for each well of a six-
well plate is 1
ʼ
g.
(b) To each reaction tube, add pre-warmed Opti-MEM
®
I
Reduced Serum Medium (volume of medium = 100
ʼ
ʼ
l,
volume of DNA to be added).
(c) Add 3
l of FuGENE
®
6 transfection reagent to the
medium (transfection reagent: DNA = 3:1), mix imme-
diately, and incubate at room temperature for 5 min
(
see
Note 20
).
(d) Add 1
ʼ
g of Myc-tagged siRNA-resistant rescue constructs
to the mixture from step (c); mix well and incubate at
room temperature for 15 min (
see
Note 21
).
9. Slowly add the 100
ʼ
l transfection reactions dropwise to each
well of the six-well using a 200-
ʼ
l micropipette tip. After 48 h,
remove the medium and wash each well three times with
2 ml PBS.
Procedures below are performed at room temperature
unless specifi ed.
10. Incubate the cells in 3.7 % formaldehyde in 200 mM HEPES
pH 7.4 for 20 min for fi xation. Remove the fi xative and wash
cells twice with PBS.
ʼ
