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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