Biology Reference
In-Depth Information
6.
Trypsin/EDTA solution: 10X stock (Gibco, Southersburg, MD, cat. no. 25090-028)
(store at -20
°
C) diluted to 1X with 1X PBS (aliquot and store at -20
°
C, store
aliquots in use at 4
°
C).
7.
Feeder medium: DMEM high glucose with Glutamax-1 (Gibco, cat. no. 61965-026)
supplemented with 10% fetal bovine serum (FBS) (Gibco, cat. no. 10270-106).
8.
Freezing medium: 70% DMEM, 20% FBS, 10% DMSO.
9.
ES medium: DMEM high glucose with Glutamax-1 (Gibco, cat. no. 61965-026)
+ Na-pyruvate (Gibco, cat. no. 11360-039) supplemented with 20% FBS (FBS
needs to be tested for ES cell use, or can be bought from Gibco already tested,
cat. no. 10270-106), 0.1 m
M
2-mercaptoethanol, 5 mL 100X nonessential amino
acids (NEA) (Gibco, cat. no. 11140-035), and 1000 U/mL leukemia inhibitory
factor (LIF) (ESGRO from Gibco, cat. no. 13275-029).
10.
Mycoplasma PCR Primer Set (Stratagene, cat. no. 302007).
2.3.2. Electroporation and Selection
1. Feeder medium (
see
Subheading 2.3.
).
2. Feeder cells (
see
Subheading 2.3.
).
3. Restriction enzymes and buffers.
4. Chloroform.
5. Phenol/chloroform: mix 1 vol of Phenol (equilibrated with 10 m
M
Tris-HCl,
1 m
M
EDTA, pH 8.0) with 1 vol of chloroform.
6.
3
M
Na-acetate pH 5.2, adjust pH with glacial acetic acid (can be stored at RT).
7.
100% ethanol.
8.
70% ethanol.
9.
Trypsin/EDTA (
see
Subheading 2.3.
).
10.
ES cells.
11.
ES medium (
see
Subheading 2.3.
).
12.
1X PBS (
see
Subheading 2.3.
).
13.
Burker's chamber.
14.
Electroporation cuvets for 0.4 cm wide (Bio-Rad, Richmond, CA).
15.
Gene Pulser™ (Bio-Rad).
2.3.3. Picking and Freezing of Resistant Clones
1.
ES medium (
see
Subheading 2.3.
).
2.
G418 (Geneticin) (Gibco or Sigma).
3.
Feeder cells (
see
Subheading 2.3.
).
4.
Feeder medium (
see
Subheading 2.3.
).
5.
24-Well plates (Falcon, Los Angeles, CA).
6.
96-Well plates (Falcon).
7.
Trypsin/EDTA (
see
Subheading 2.3.
).
8.
Stereomicroscope.
9.
Cryovials (Sarstedt, Germany).
10.
Cryovial rack (Sarstedt).
11.
1X PBS (
see
Subheading 2.3.
).
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