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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