Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 146

3. Sterile DDW.

4. Taq DNA polymerase (for example, Gibco, cat. no. 10342-012).

5. Agarose and ethidium bromide.

2.7. Generation and Analysis of Double KO ES Cells
1. ES cells.

2. ES medium ( see Subheading 2.3. ).

3. Feeder cells ( see Subheading 2.3. ).

4. 9-cm tissue-culture dishes (Falcon).

5. G418 (Geneticin).

2.7.1. Analysis of ES Contribution in Chimeric Mice

1. Surgical equipment ( see Subheading 2.8. ).

2. Extraction buffer: 50 m M Tris pH 8.0 0.1% Triton.

3. Disposable plastic pestles (Eppendorf, Germany).

4. Titan III cellulose acetate plate (HELENA Laboratories, Beaumont, TX, cat. no. 3033)

5. Super Z Applicator Kit (HELENA Laboratories, cat. no. 4093) containing Super

Z applicator, Super Z aligning base, Super Z well plate.

6. Paper towels and Whatmann 3M filter paper.

7. Supre Heme buffer (HELENA Laboratories, cat. no. 5802).

8. Electrophoresis chamber.

9. Glass slides.

10. 1% agarose in 0.2 M Tris-HCL, pH 8.0.

11. Stock solutions for staining. Keep in 200- L aliquots at indicated temperatures.

• Magnesium acetate 0.25 M . Store at -20

µ

C.
• Fructose-6-Phosphate (Sigma, cat. no. F3627) 75 mg/mL. Store at -20

°

° C.

• Tetrazolium (MTT) (Sigma, cat. no. M2128) 10 mg/mL. Store at -20 C.

•Phenazine methosulfate (PMS) (Sigma, cat. no. P9625) 1.8 mg/mL. Store
at -20

°

C in the dark.
• Nicotinamide adenine dinucleotide phosphate (NADP) (Sigma, cat. no.
N0505). Store at -20

°

C.
• Glucose-6-phosphate dehydrogenase (G6PDH) (Sigma, cat. no. G8878). Store
at +4

°

° C.

12. Stop solution: 5% acetic acid.

2.8. Analysis of Differentiation Abilities
of Homozygous ES Cells in Embryo Bodies and Teratomas
2.8.1. Generation of ES Cells-Derived Embryoid Bodies

1. ES cells.

2. ES medium ( see Subheading 2.3. ).

3. Feeder cells ( see Subheading 2.3. ).

4. Feeder medium ( see Subheading 2.3. ).

5. 1X PBS ( see Subheading 2.3. ).