Danilchicks Medium for Amy (DFA):



What

Stock

in 500 ml

final Conc.





NaCl 4M NaCl 6.625ml 53 mM
Na2CO3 1M Na2CO3 2.5 ml 5 mM
Potasium Gluconate (e.g. Sigma G4500) powder 0.525 grams 4.5 mM




Sodium Gluconate (e.g. Sigma G9005) powder 3.49 grams 32 mM




CaCl2 1 M CaCl2 1 ml 1 mM
MgSO4 1 M MgSO4 1 ml 1 mM


Make up in ultrapure filtered 18 M Ohm-cm water (e.g. Nanopure, milliQ) or molecular biology grade "box" water

pH to 8.3 with 1 M Bicine

add 0.1% BSA  (0.5 grams/ 500 mls)

bring to final volume

Sterile filter (0.2 micron) into an autoclaved (or otherwise sterile) bottle.

Aliquot 40 mls into 50 ml conical tubes and store at -20C.

To use, we thaw these tubes, usually in water (some think hot water is bad for them). 

Once thawed and at about room temp, we add 400 microliters of Antibiotic/antimycotic 100X solution (Sigma A5955).  This stuff seems to go off, and in fact become toxic fairly fast if left at room temp for any length of time.
Thaw the bottle in cold water and aliquot immediately. Good for culture for about 1-2 days.

We routinely culture explants in DFA.  We do transplant and graft operations in DFA and then transfer to 1/3X MBS once they've healed (1-2 hours).

Lab wisdom has it that embryos cultured in 1/3X MBS are easier to operate on than those culture in more dilute salines.

Embryos that have been cultured below about 15C tend to be mushy or sickly and are often very difficult to work on.  Even embryos that have been  cultured at about 15 to 17 tend to be a bit mushy, but we routinely use these temps anyway, for convenience.