Biology Reference
In-Depth Information
12.
Freezing medium (
see
Subheading 2.3.
).
13.
Dry ice.
2.3.4. Identification of Homologous Recombinants
1.
Lysis buffer: 100 m
M
Tris-HCL, pH 8.5, 5 m
M
EDTA, 0.2% SDS, 200 m
M
NaCl, 100
g/mL proteinase K (Sigma, USA, P0390). Keep proteinase K stock
solution (10 mg/mL) at -20
µ
°
C and always add freshly.
2.
Isopropylic alcohol.
3.
Restriction enzymes and buffers.
4.
DNase-free bovine serum albumin (BSA) (New England Biolabs, Beverly, MA).
5.
Agarose and ethidium bromide.
6.
10X TBE-buffer: 108 g Tris-base, 55 g boric acid, 9 mL 0.5
M
EDTA pH 8.0,
adjust to 1 L with deionized water (can be stored at RT).
7.
Nylon membrane (
see
Subheading 2.1.
).
8.
Denaturation solution (
see
Subheading 2.1.
).
9.
SSC 20X (
see
Subheading 2.1.
).
10.
Hybridization plastic bags.
11.
Church buffer (
see
Subheading 2.1.
).
12.
Random priming labeling kit for DNA
see
Subheading 2.1.
).
32
P-CTP (
see
Subheading 2.1.
).
13.
14.
Wash solution 1 (
see
Subheading 2.1.
).
15.
Wash solution 2 (
see
Subheading 2.1.
).
16.
Autoradiography film.
2.3.5. Transient Cre Transfection
1.
ES cells.
2.
ES medium (
see
Subheading 2.3.
).
3.
6-Wells microtiter dishes.
4.
9-cm tissue-culture dishes.
5.
pIC-Cre plasmid
(5)
.
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 solution (
see
Subheading 2.3.
).
10.
Gancyclovir (Syntex, cat. no. 00033-2903-48).
2.4. Generation of Mutant Mouse Lines
2.4.1. Generation of Vasectomized Males
1.
Mice for vasectomy: 8- or more wk-old FVB males.
2.
Avertin 100% stock: Dissolve 10 g 2,2,2-tribromoethyl alcohol (Fluka, Switzer-
land, cat. no. 90710) in 10 mL tert-amyl alcohol. For use, dilute the stock solu-
tion to 2.5% in PBS. Store both stock and use solutions at 4
°
C wrapped in
aluminum foil to protect them from light.
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