Hybridization Buffer

1. Stock solutions.

a. 20X SSC: 3M NaCl, and 0.3M sodium citrate, pH 7.0. Treat with DEPC (0.1% [v/v]) and autoclave.

b. Denhardt's solution (50X stock): Dissolve 100 mg each of Ficoll, polyvinylpyrrolidone, and BSA in 10 mL of DEPC-treated water, filter through a 0.2-pm filter into autoclaved microfuge tubes, and store at -20°C in 400-|nl aliquots.

c. 1M DTT in 0.1M sodium acetate: Filter through 0.2-|im filter into autoclaved microfuge tubes, and store at -20°C in 100-pL aliquots.

d. Yeast tRNA (Sigma) in DEPC-treated water: Filter through 0.2-pm filter. Store at -20°C in 100-pL aliquots.

e. Sheared salmon sperm (ss) DNA (Sigma): Dissolve at 10 mg/mL in DEPC-treated water Heat in a boiling water bath for 10 min.

f. 50% (v/v) deionized formamide (Fluka, Buchs, Switzerland).

g. Dextran sulfate (500,000 mol wt).

2. Hybridization buffer (20 mL). Mix the following:

Amount Final conc.

Deionized formamide lOmL 50% (v/v)

20X SSC 4 mL 4X

Sheared ssDNA 10 mg/mL 1 mL 500 pg/mL

Yeast tRNA 0.1 mL 5 mg/mL

5OX Denhardt's sol'n 0.4 mL IX

*Vortex to dissolve

This solution can be stored at -20°C for at least 2 mo. Store in single-use aliquot (e.g., 1 mL) in autoclaved microfuge tubes. To use with a 35S-labeled probe, add 10 pL 1M DTT/mL of hybridization buffer prior to use.

3 Oligonucleotide probe diluted to a concentration of 100 ng/mL.

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