Biology Reference
In-Depth Information
(Narishige, Japan). Two 10-mL syringes, each linked to a metal glass capillary
holder (Narishige, Japan) via a silicon tube.
2.
Injection chamber: lid of a 3-cm tissue-culture dish with a hole in the middle
(about 1 cm in diameter).
3.
Vaseline without any additives.
4.
Siliconized coverslip: rinse the coverslips in chloroform 2% dimetildiclorosilane
for 30 s and air-dry.
5.
M2 medium: 94.66 m M NaCl, 4.78 m M KCl, 1.71 m M CaCl 2 , 1.19 m M KH 2 PO 4 ,
1.19 m M MgSO 4 , 4.15 m M NaHCO 3 , 20.85 m M HEPES, 23.28 m M sodium lac-
tate, 0.33 m M sodium pyruvate, 5.56 m M glucose, BSA 4 g/L.
6.
Petri dish (Falcon, Los Angeles, CA).
7.
Ice.
8.
Dimethyl polysiloxan (Sigma, cat. no. 9016-00-6).
2.4.7. Embryo Transfer
1.
Microinjected blastocysts.
2.
Pseudopregnant female mouse.
3.
Avertin ( see Subheading 2.8. ).
4.
Two stereomicroscopes.
5.
Optic fibers illuminators.
6.
Surgical equipment ( see Subheading 2.8. ).
7.
Transfer glass pipet.
2.5. Mating of Chimeras
1.
Adult chimeric males.
2.
C57B6 females.
3.
Adult 129 females.
2.6. Genotyping Offspring
2.6.1. Southern Blot Analysis of Tail DNA
1.
20-30-d-old mice.
2.
Ear-clips (National Band and Tag Co.).
3.
Rotary wheel.
4.
Tail buffer-PK: 1 m M Tris-HCl, pH 7.5, 1 m M EDTA, 250 m M NaCl, 0.2% SDS
and 0.1 mg/mL of freshly added Proteinase K (Sigma, cat. no. P0390).
5.
Phenol/chloroform ( see Subheading 2.4. ).
6.
Chloroform.
7.
Isopropyl alcohol.
8.
Sterile DDW.
2.6.2. DNA Preparation for PCR Analysis
1.
Lysis buffer: PCR buffer X1 and 0.1 mg/mL proteinase K in DDW.
2.
Thermomixer.
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