Biomedical Engineering Reference
In-Depth Information
3 Place the plate in the incubator for 5min. Rock the cells once during this time to
promote dissociation from the plate.
4 Add 9 ml of ES medium and rock gently.
5 Dissociate cells by pipetting up and down 15 times while forcing the pipette tip
against the bottom of the dish.
o
6 Transfer the cells to a 50ml conical tube and spin for 3min at 600
g
.
7 Aspirate the supernatant and thoroughly resuspend cells in 10ml of PBS.
8Dilute5
μ
lofcellsuspensionin20
μ
l of trypan blue (1 : 5 dilution). Add to a
hemacytometer and calculate the number of cells per milliliter.
9 For each electroporation, spin down 20 million cells and resuspend the pellet in PBS
with 25
μ
g of linearized targeting vector DNA in a total volume of 800
μ
l.
10 Add resuspended cells to a 4mm sterile, disposable gap cuvette (800
μ
l
capacity).
11 Cap cuvette and let stand for 5 min at room temperature.
12 Apply a single 225 V, 500
μ
F pulse to each cuvette (ECM 630 - low-voltage mode,
resistance
=
0). Let the cuvette stand on ice for 10min.
13 Transfer cells to 9.2ml of ES cell medium in a 15ml conical tube. Rinse cuvette with
medium to maximize recovery of cells.
14 Take 100
μ
l of the 10ml of electroporated cells and calculate survival rate: mix 100
μ
l
of cells with 100
μ
l of trypan blue, wait 5min and count number of live cells. Survival
rate equals number of live cells after electroporation divided by 20 million. A typical
electroporation will have a survival ratio 30-50%.
15 Prepare
γ
-MEF cells plates by substituting MEF medium with ES medium. Typically,
each electroporation will seed five plates.
16 Plate 2 ml of cells (from 10ml) in each plate and disperse to allow good separation
between colonies.
17 After 24 h, change medium from ES medium to ES medium
+
2
×
G418 (1 : 83.5 dilution
of G418).
p
18 48 h after electroporation, change medium to ES medium
+
1
×
G418
+
FIAU.
q
19 Change medium once per day with ES medium
+
1
×
G418
+
FIAU.
Notes
n
Cells will be ready for electroporation approximately four days after being passaged. Each plate
typically yields 10-20 million cells.
o
It is critical that a thorough dissociation be accomplished, so as to allow for efficient
electroporation and subsequent plating of individual cells.
p
ES cells will grow on days 2-6 after electroporation, but most will die off as the cells only
transiently transfected are eliminated after gradual loss of the neomycin resistance cassette.
By days 7-9, individual colonies should become visible on the surface of the MEF cells and be
ready for picking.
