Solutions

All ingredients are made from RNase-free solutions. Refer to any standard textbook on molecular biology for details on the preparation of the stock solutions required (30). Sodium-heparin is purchased from Sigma Chemicals, Inc., dissolved in DEP-treated water, and stored in 0.5 ml aliquots at -20 °C.

Make the pre-hybridization/hybridization solution fresh each time, preparing enough solution for both the pre-hybridization and hybridization. After pre-warming to 42 °C, remove half for hybridization, and store at 4°C until ready to perform the hybridization (if doing overnight pre-hybridization), then pre-warm to 42°C again. The denatured DNA should be added to both the pre-hybridization and hybridization solution just before use.

Sheared or sonicated salmon sperm DNA at 10 mg/ml in TE buffer is boiled for 10 min, quick-cooled on ice for 5 min, and 0.25 ml added per 10 ml.

DNA probes are radiolabeled by the random primer method, purified by centrifugation through Sephadex G50, and boiled in TE buffer for 10 min,

Per 50 ml

Formamide (deionized) 1 M Tris-base (pH 7.4) 5 M NaCl

50% dextran sulfate 10% SDS

50 X Denhardt's solution 50 mg/ml Na-heparin

25 ml 2.5 ml 10 ml 10 ml 5 ml lml 0.5 ml then cooled on ice for 5 min before addition to hybridization buffer. The unused probe can be stored at -20°C and used for up to 2 weeks in many cases. The specific activity of probes should be ~109 cpm/(xg.

Was this article helpful?

0 0

Post a comment