Gal Staining Colonies

1. Phosphate buffer (0.1 M) made by adding 21 parts of 0.1 M Na2HPO4 (Sigma) to 4 parts 0.1 M NaH2PO4 (Sigma) to give a final pH of 7.5.

2. 5-Bromo-4-chloro-3-indolyl-P-d-galactopyranoside (X-gal, Boerhinger-Mannheim) stock solution, 50 mg/mL, dissolved in dimethyl formamide (Sigma). Store in the dark at -20°C.

3. Fix buffer: 0.1 M Phosphate buffer, pH 7.3, 2 mM MgCl2, 0.2% Gluteraldehyde (Sigma), 0.5 mM EGTA (pH 8.0), Store at 4°C for up to 1 mo.

4. Wash buffer: 0.1 M phosphate buffer, 2 mM MgCl2, 0.1% sodium deoxycholate (Sigma), 0.02% Nonidet P-40 (Sigma). Store at 4°C.

5. X-gal staining solution:

0.106 g Potassium ferrocyanide (Sigma).

0.082 g Potassium ferricyanide (Sigma).

1 mL X-gal (50 mg/mL). Filter to remove crystals, and store in the dark at 4°C. Add to 50 mL of X-gal wash buffer.

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