Culture Media

1. Embryos and explants are cultured in dilutions of Normal Amphibian Medium (NAM; 14): 110 mM NaCl; 2 mM KCl; 1 mM Ca(NO3)2; 1 mM MgSO4; 0.1 mM Na2EDTA; 2 mM sodium phosphate pH 7.5; 1 mM NaHCO3; 50 pg/mL gentamycin. It is convenient to prepare a 10X stock solution of all the components of NAM with the exception of the phosphate buffer, the NaHCO3, and the gentamycin. This 10X 'NAM salts' stock may be autoclaved. We then prepare:

a. 0.1 M Sodium phosphate pH 7.5, which may be autoclaved and serves as a 50X stock.

b. 0.1 M NaHCO3, which is filter-sterilized and stored frozen and serves as a 100X stock.

c. 10 mg/mL gentamycin which is also filter-sterilized and stored frozen and serves as a 200X stock.

Dilutions of NAM refer only to the "NAM stocks" and NaHCO3; levels of phosphate buffer and gentamycin are held constant.

4. 60% Leibovitz L-15 medium (for storage of Xenopus testes).

5. 2% Cysteine hydrochloride, adjusted to pH 7.9-8.1 with NaOH.

6. Sterile mineral oil.

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