M9 minimal medium - Revision history

Jstuelk at 18:02, 7 July 2013

2013-07-07T18:02:32Z

Jstuelk at 18:01, 7 July 2013

2013-07-07T18:01:37Z

Jstuelk at 17:10, 7 July 2013

2013-07-07T17:10:37Z

Jstuelk at 13:18, 4 July 2013

2013-07-04T13:18:13Z

Jstuelk: Created page with "This medium is used to grow ''B. subtilis'' in the presence of glucose or other variable carbon sources. The medium does contain ammonium and is therefore not suited to change..."

2013-07-04T13:15:39Z

Created page with "This medium is used to grow ''B. subtilis'' in the presence of glucose or other variable carbon sources. The medium does contain ammonium and is therefore not suited to change..."

New page

This medium is used to grow ''B. subtilis'' in the presence of glucose or other variable carbon sources. The medium does contain ammonium and is therefore not suited to change the nitrogen source.

== The basal solutions ==
* 5x M9 stock solution, per liter
** 42.5 g Na2HPO4.2H2O (Merck)
** 15 g KH2PO4 (Merck)
** 5.0 g NH4Cl (Merck)
** 2.5 g NaCl (Merck)

** Adjust to pH 7.0 using 4M NaOH. Solution is autoclavable at 121oC for 20 minutes. Store at RT.

* Trace elements solutions, 100x stock solution, per liter
** 100 mg MnCl2.4H2O (Merck)
** 170 mg ZnCl2 (Sigma)
** 43 mg CuCl2.2H2O (Merck)
** 60 mg CoCl2.6H2O (Merck)
** 60 mg Na2MoO4.2H2O (Aldrich)

** Contrary to what is described by Harwood and Cutting, MgSO4 and CaCl2 are added as separate solutions to prevent precipitation problems. FeCl3 is also added as a separate solution as this compound is unstable. Filter-sterilize the solution and store in the dark at RT.

* Calciumchloride solution (100mM, per 100 mL):
** 1.47 g CaCl2.2H2O (Merck)
** Autoclave and store at RT.

* Magnesiumsulfate solution (1M, per 100 mL):
** 24.6 g MgSO4.7H2O (Merck)
** Autoclave and store at RT.

* Iron chloride solution (50 mM in 100 mM citric acid, 100 mL):
** 1.35 g FeCl3.6H2O (Sigma)
** 2.10 g citric acid.H2O (Sigma)
** Filter sterilize and store in the dark at 4oC.

* Glucose solution:
** 500 g C6H12O6 (Merck)
(50% w/v, per liter)
Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution is autoclavable at 121oC for 20 minutes. Filter sterilization is also possible.

Or H) Glucose solution: 200 g C6H12O6 (Merck)
(20% w/v, per liter)
Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution should be filter sterilized.

I) L-malate solution: 134 g C4H6O5 (Sigma) L-form!!
(13.4% w/v, per liter)
Use a 4M NaOH solution to adjust the pH of the solution to neutral (pH 6.0-8.0). Requires roughly 47ml 4M NaOH per 100ml of solution. Solution should be filter sterilized.

NOTE: For all solutions Nanopure H2O with a electric resistance of more than 17 MOhm/cm (= conductivity of less than 60 nS * cm) is used.

== Preparation of the medium ==
* For 1 liter of M9 medium add in the following order:
** 200 mL M9 5x stock (sol. C)
** 1 mL 100 mM CaCl2 (sol. E)
** 10 mL trace salts 100x stock (sol.D)
** 1 mL 1 M MgSO4 (sol. F)
** 1 mL 50 mM FeCl3/100 mM C6H8O7 (sol. G)
** 6 mL of glucose 50% (w/v) stock solution (sol. I)

** Or 15 mL of glucose 20 % or 15 mL of DL-malate 20% for 0.3 % carbone source
** Or 10 mL glucose 50%, or 25 mL glucose 20%, or 25 mL DL-malate 20% for 0.5%

** add nanopure H2O to a final volume of 1 liter

== Back to [[Methods]] ==

@@ Line 56: / Line 56: @@
 ** 6 mL of glucose 50% (w/v) stock solution (sol. I)
-** Or  15 mL of glucose 20 % or 15 mL of DL-malate 20% for 0.3 % carbone source
+** Or  15 mL of glucose 20 % or 15 mL of DL-malate 20% for 0.3 % carbon source
 ** Or 10 mL glucose 50%, or 25 mL glucose 20%, or 25 mL DL-malate 20% for 0.5%
@@ Line 62: / Line 62: @@
 == Note ==
-* This medium was used for LCA experiments (using strains derived from the Trp+ BSB168 strain) in the BaSysBio consortium. It is not
+* This medium was used for LCA experiments (using strains derived from the Trp+ BSB168 strain) in the BaSysBio consortium. It is not supplemented with Trp (in order to minimize the fluorescence of the medium when monitoring GFP production). However, even when adding Trp, the Trp minus strains grow usually pretty bad in this medium.
 == Reference ==
   '''Harwood, C. R., and S. M. Cutting.''' 1990. Chemically defined growth media

@@ Line 61: / Line 61: @@
 ** add nanopure H2O to a final volume of 1 liter
 == Reference ==
   '''Harwood, C. R., and S. M. Cutting.''' 1990. Chemically defined growth media

← Older revision		Revision as of 17:10, 7 July 2013
Line 61:		Line 61:
	** add nanopure H2O to a final volume of 1 liter		** add nanopure H2O to a final volume of 1 liter

		+	== Reference ==
		+	'''Harwood, C. R., and S. M. Cutting.''' 1990. Chemically defined growth media
		+	and supplements, p. 548. In C. R. Harwood and S. M. Cutting (ed.), Molecular
		+	biological methods for Bacillus. Wiley, Chichester, United Kingdom.

	== Back to [[Methods]] ==		== Back to [[Methods]] ==

@@ Line 32: / Line 32: @@
 ** Filter sterilize and store in the dark at 4oC.
-* Glucose solution:
+* Glucose solution (50% w/v, per liter)	:
-** 500 g	C6H12O6 (Merck)
+** 500 g	glucose (Merck)
-	(50% w/v, per liter)
+** Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution is autoclavable at 121oC for 20 minutes. Filter sterilization is also possible.
-Or H) Glucose solution:	200 g	C6H12O6 (Merck)
+* Glucose solution (20% w/v, per liter):
-	(20% w/v, per liter)
+** 200 g	C6H12O6 (Merck)
-Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution should be filter sterilized.
+** Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution should be filter sterilized.
-I) L-malate solution:	134 g	C4H6O5 (Sigma) L-form!!
+* L-malate solution (13.4% w/v, per liter):
-	(13.4% w/v, per liter)
+** 134 g	L-malate (Sigma)
-Use a 4M NaOH solution to adjust the pH of the solution to neutral (pH 6.0-8.0). Requires roughly 47ml 4M NaOH per 100ml of solution. Solution should be filter sterilized.
+** Use a 4M NaOH solution to adjust the pH of the solution to neutral (pH 6.0-8.0). Requires roughly 47ml 4M NaOH per 100ml of solution. Solution should be filter sterilized.
-NOTE: For all solutions Nanopure H2O with a electric resistance of more than 17 MOhm/cm (= conductivity of less than 60 nS * cm) is used.
+=== NOTE: For all solutions Nanopure H2O with a electric resistance of more than 17 MOhm/cm (= conductivity of less than 60 nS * cm) is used ===