Benson Lab

Frankia Defined Minimal Medium (FDM)

For routine growth and subculture of Frankia in 500 ml screw-capped flasks.

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Component	Per liter
NaSuccinate (CpI1)	5 g
Fructose (EanI1)	5 g
NaPyruvate (CcI3)	5 g
NH4Cl	0.5 g
20X stock salts*	50 ml
1000X Iron stock**	1 ml
10X Buffer stock***	100 ml (adjust pH to 6.5; add after autoclaving)

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To make 1 liter of medium, start with 850 ml dH₂O, add carbon source and ammonium chloride, 50 ml of the 20X stock salt solution, 1 ml of iron solution and distribute 180 ml each to 500 ml Erlenmeyer flasks.  Autoclave the phosphate buffer separately and add 20 ml to each flask after cooling.

*20X Stock Salts: CaCl₂.2H₂O, 2.0 g; MgSO₄.7H₂O, 4.0 g; dH₂O, 1000 ml

**1000X Stock Iron: Dissolve 0.75 g Na₂EDTA.2H₂O, 0.56 g FeSO₄.7H₂O and 0.02 g of Na₂MoO₄.2H₂O in 100 ml dH₂O

***10X Buffer: KH₂PO₄, 68.0 g/l; K₂HPO₄.3H₂O, 114.1 g/l.  Separately make up one 1 l of monobasic plus 500 ml of dibasic, add dibasic to monobasic slowly to adjust pH to 6.5.

Notes:

- Proteose Peptone #3 can be added at a level of 0.5 g/l for some growth improvement.

- Although more expensive, MOPS can be substituted for some of the phosphate buffer especially in nitrogen-fixing cultures.  In that case, make up a 10X NaMOPS solution to 200 mM, pH6.5 by adding 46 g to 1 liter dH₂O and then replacing some of the 10X phosphate. Lower the phosphate to 1 mM (by adding 1.0 ml instead of 20 ml of the 10X P-stock)