Biology Reference
In-Depth Information
2.
Freezing solution for ES- and EF-cell storage: 10% DMSO (Sigma; #D2650) in
standard ES-cell medium; the freezing solution should be made fresh each
time is used.
3.
Trypsin: 0.25% trypsin/1 m
M
EDTA in Hepes-buffered saline (Gibco; #25200-056).
4.
Gelatin (Fisher; #G8-500): 0.2% in ultrapure water, autoclaved and stored at room
temperature.
2.2. ES-Cell Electroporation
1.
Electroporation buffer: 20 m
M
Hepes pH 7.0, 137 m
M
NaCl, 5 m
M
KCl, 0.7 m
M
Na
2
HPO
4
, 6 m
M
glucose, 0.1 m
M
-mercaptoethanol. Each stock solution should
be autoclaved with the exception of glucose which should be sterilized through a
0.22-
β
m pore size filter. Once prepared, the electroporation buffer is again steril-
ized through a 0.22-
µ
µ
m pore size filter before being stored at 4
°
C.
2.
Selection buffer: Same as the ES medium, except that G418 (Gibco; #11811-031)
is added at a final concentration of 350
µ
g/mL. Make a 500X stock solution in
H
2
O and store it at -20
C. Because G418 activity can vary from batch to batch, it
is best to determine the minimal concentration required to kill 100% of ES cells
for each batch.
°
3.
Electroporation set-up: Bio-Rad Gene Pulser™ and cuvettes (Bio-Rad, Rich-
mond, CA; #165-2088).
2.3. ES-Clone Analysis
1.
96-Well V-bottom plates (Costar; #3894), 24-well plates (Costar, Cambridge,
MA; #3524), 96-well flat-bottom plates (Costar;#3599).
2.
Multichannel pipet (Costar; #4880).
3.
Pipet tips from USA Scientific (#10110006) are especially well-suited for pick-
ing up ES colonies.
4.
DNA lysis buffer: 100 m
M
Tris-HCl pH 8.5, 5 m
M
EDTA, 0.2% SDS, 200 m
M
NaCl, 100
µ
g/mL Proteinase K (Sigma; #P2308).
2.4. Blastocyst Microinjection
1.
Microinjection set-up,
see
Subheading 3.5., step 1.
2.
M2 medium (Sigma; #M5910).
3.
M16 medium (Sigma; #M7292).
3. Methods
3.1. ES-Cell Cultures
1.
C in a CO
2
incubator with
daily changes of ES medium and on a feeder layer of G418
r
EF cells in gelati-
nized tissue culture plates or flasks
(6)
. Frozen vials of ES or EF cells are thawed
as quickly as possible at 37
Seeding ES cells: ES cells are always grown at 37
°
C and then sterilized with ethanol. To remove the
cytotoxic DMSO, add the cell suspension to a sterile conical tube filled with ~10
times excess of ES medium. Pellet the cells at room temperature and resuspend
°
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