Chlamy Growth Media

Adapted from various in-lab sources

Stock Solutions

	Component	% chemical in stock	in 1L stock
#1	Trace Elements	Look Right	-
#2	Na Citrate·2H₂O	10%	100g
#3	FeCl₃·6H₂O	1%	10g
#4	CaCl₂·6H₂O	5.3%	53
#5	MgSO₄·7H₂O	10%	100g
#6	NH₄NO₃	10%	100g
#7	KH₂PO₄	10%	100g
#8	K₂HPO₄	10%	100g

Trace Elements
Component	in 1L stock
H₃BO₃	1g
ZnSO₄·7H₂O	1g
MnSO₄·H₂O	0.303g
CoCl₂·6H₂O	0.2g
Na₂MoO₄·2H₂O	0.2g
CuSO₄	0.04g

Recipes

For 1L of media

Component	M	M/NO₃	M - N	R	R/NO₃	R - N	SG II	SG II/NO₃
#1	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml
#2	5 ml	5 ml	5 ml	5 ml	5 ml	5 ml	5 ml	5 ml
#3	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml
#4	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml	1 ml
#5	3 ml	3 ml	3 ml	3 ml	3 ml	3 ml	3 ml	3 ml
#6	3 ml	-	-	4 ml	-	-	3 ml	3 ml
#7	1 ml	0.8 ml	-	2 ml	1 ml	-	-	-
#8	1 ml	1.5 ml	2 ml	3.5 ml	1.75 ml	3.5 ml	-	-
1M KNO₃	-	4 ml	-	-	4 ml	-	-	4 ml
2.2M Na Acetate	-	-	-	6 ml	6 ml	6 ml	-	-
K₂HPO₄	-	-	-	-	-	-	1.5 g	1.5 g
NaH₂PO₄·H₂O	-	-	-	-	-	-	3.67 g	3.67 g
Na Acetate·3H₂O	-	-	-	-	-	-	2 g	2 g
For arginine requiring mutants add 2ml of 10% arginine to 1L of media.

TAP Media

Recipe

Stock Solution	For 1 L
1M Tris base	20 ml
Phosphate Buffer II	1 ml
Hutner's Trace Metals	1 ml
Solution A	10 ml
Glacial acetic acid (pH to 7.0)	1 ml

Stock Solutions

Phosphate Buffer II
Component	For 100 ml
K₂HPO₄	10.8 g
KH₂PO₄	5.6 g

Solution A
Component	for 500 ml
NH₄Cl	20 g
MgSO₄·7H₂O	5 g
CaCl₂·2H₂O	2.5 g

Hutner's Trace Metals

Dissolve 50g of acid free EDTA in 250 ml of ddH₂O. Heat to dissolve.

Dissolve the following, one by one, in order, heating to approximately 100° in 550 ml ddH₂O.

BO₃H₃	11.4 g
ZnSO₄·7H₂O	22.0 g
MnCl₂·4H₂O	5.06 g
FeSO₄·7H₂O	4.99 g
CoCl₂·6H₂O	1.61 g
CuSO₄·5H₂O	1.57 g
Mo₇O₂₄(NH₄)₆·4H₂O (Ammonium Molybdate)	1.1 g

Mix the two solutions together. The resulting solution should be blue-green.

Heat to 100°. Cool slightly, but don't let the temperature drop below 80°-90°.

Adjust pH to 6.5 to 6.8 with 20% KOH (approximately 83 ml). Don't let the temperature drop below 70 ° until after the pH is adjusted.

Make up to 1L and let stand in a 2L Erlenmeyer flask, stoppered loosly. The colour should change from green to purple.

Remove the rust-coloured precipitate by filtering, with suction, through 3 layers of Whatman #1 filter paper in a Buchner funnel. Repeat until no more precipitate is seen on the filter paper.

Store in a brown bottle at 4°.

Gorman, D.S. and R.P. Levine (1965). Proc. Natl. Acad. Sci. USA 54:1665-1669.
Hutner, et al. (1950). Methods in Enzymology, 23:68.
Sager, R. and S. Granick (1953). Ann. N.Y. Acad. Sci. 56:831-838.