Media preparation - Revision history

Mjeppesen at 20:34, 21 July 2022

2022-07-21T20:34:38Z

← Older revision		Revision as of 20:34, 21 July 2022
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	~~[[Category:Protocols]]~~
	[[Category:Protein expression]]		[[Category:Protein expression]]
	[[category:Cell_Culturing]]		[[category:Cell_Culturing]]

Mjeppesen: Added to Category

2021-09-11T05:05:11Z

Added to Category

← Older revision		Revision as of 05:05, 11 September 2021
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	[[Category:Protocols]]		[[Category:Protocols]]
	[[Category:Protein expression]]		[[Category:Protein expression]]
			[[category:Cell_Culturing]]
	===1. General Protocols For Minimal Media Protein Expression===		===1. General Protocols For Minimal Media Protein Expression===

Eriekeberg: Formatting Only

2015-07-15T14:23:07Z

Formatting Only

← Older revision		Revision as of 14:23, 15 July 2015
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	64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O		64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O

	15 g KH<sub>2</sub>PO<sub>4</sub>		15 g KH<sub>2</sub>PO<sub>4</sub>

	2.5 g NaCl		2.5 g NaCl

Bozhang at 20:45, 3 September 2013

2013-09-03T20:45:52Z

← Older revision		Revision as of 20:45, 3 September 2013
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	[[Category:Protocols]]		[[Category:Protocols]]
	[[Category:Protein expression]]		[[Category:Protein expression]]

	===1. General Protocols For Minimal Media Protein Expression===		===1. General Protocols For Minimal Media Protein Expression===

Bozhang at 19:43, 3 September 2013

2013-09-03T19:43:52Z

← Older revision		Revision as of 19:43, 3 September 2013
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	[[Category:Protocols]]		[[Category:Protocols]]
	[[Category:Protein expression]]		[[Category:Protein expression]]
	~~[[Category:Metabolomics]]~~

	1. General Protocols For Minimal Media Protein Expression		===1. General Protocols For Minimal Media Protein Expression===

	Expression Protocol in M9 Minimal Media via T7 Promoter: The following protocol has been used successfully to <sup>15</sup>N or <sup>13</sup>C/<sup>15</sup>N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus ~~NH4Cl~~. Standard 5 X M9 Minimal Media salts minus nitrogen source ~~For~~ 1L 5xM9 salts:		Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH<sub>4</sub>Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source for 1L 5xM9 salts:

	64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O		64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O
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	To prepare 500 mL M9 minimal media:		To prepare 500 mL M9 minimal media:

	100mL of 5xM9 salts		100mL of 5xM9 salts:

	1 mL 1 M MgSO<sub>4</sub>		1 mL 1 M MgSO<sub>4</sub>

	50 uL 1 M CaCl<sub>2</sub>		50 uL 1 M CaCl<sub>2</sub>

	5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)		5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
	2.5 mL filter sterilized NH<sub>4</sub>Cl (0.2 g/mL) or 0.5 g dry
	10 mL 20% d-glucose or 2 g dry		2.5 mL filter sterilized NH<sub>4</sub>Cl (0.2 g/mL) or 0.5 g dry 10 mL 20% d-glucose or 2 g dry

	Glass distilled & autoclaved H<sub>2</sub>O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).		Glass distilled & autoclaved H<sub>2</sub>O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).

	Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/ml (or any antibiotics used to select the strains).		Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/mL (or any antibiotics used to select the strains).

	Grow 5mL overnight culture in same media to inoculate 500mL M9.		Grow 5mL overnight culture in same media to inoculate 500mL M9.
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	2. General Protocols For LB Media		===2. General Protocols For LB Media===

			For 1 L media preparation

	~~For 1 L media preparation,~~
	10 g Bacto-tryptone		10 g Bacto-tryptone

	5 g yeast extract		5 g yeast extract

	10 g NaCl		10 g NaCl

	Sterilized by autoclave		Sterilized by autoclave

			Add any antibiotics used to select the strains properly after the medium temperature is blew 37 <sup>o</sup>C.

			===3. General Protocols For High-Cell-Density Media===

			For 1 L media preparation

			50 mM Na<sub>2</sub>HPO<sub>4</sub>.7H<sub>2</sub>O

			25 mM KH<sub>2</sub>PO<sub>4</sub> (pH 8.0-8.2)

			10 mM NaCl

			5 mM MgSO<sub>4</sub>

			0.2 mM CaCl<sub>2</sub>

			0.1% NH<sub>4</sub>Cl or <sup>15</sup>NH<sub>4</sub>Cl

			1.0% Glucose or <sup>13</sup>C-Glucose

			To prepare the media, glucose and metal solution are filtered to sterilize. The salt solution is autoclaved and the cooled solution is added by metal solution and glucose.

Bozhang at 04:03, 23 December 2012

2012-12-23T04:03:27Z

← Older revision		Revision as of 04:03, 23 December 2012
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	2. General Protocols For LS Media		2. General Protocols For LB Media

	For 1 L media preparation,		For 1 L media preparation,

Bozhang at 04:03, 23 December 2012

2012-12-23T04:03:06Z

← Older revision		Revision as of 04:03, 23 December 2012
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	[[Category:Metabolomics]]		[[Category:Metabolomics]]

	1. General Protocols For Minimal Media Protein Expression		1. General Protocols For Minimal Media Protein Expression
	Expression Protocol in M9 Minimal Media via T7 Promoter:The following protocol has been used successfully to ~~{{SimpleNuclide2\|~~N\| 15~~}} or 13C~~/~~15N~~ label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:
			Expression Protocol in M9 Minimal Media via T7 Promoter: The following protocol has been used successfully to <sup>15</sup>N or <sup>13</sup>C/<sup>15</sup>N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:

	64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O		64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O
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	2.5 g NaCl		2.5 g NaCl

	~~H2O~~ to final 1L volume and autoclave		H<sub>2</sub>O to final 1L volume and autoclave
	To prepare ~~500mL~~ M9 minimal media:		To prepare 500 mL M9 minimal media:

	100mL of 5xM9 salts		100mL of 5xM9 salts
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	50 uL 1 M CaCl<sub>2</sub>		50 uL 1 M CaCl<sub>2</sub>
	5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)		5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
	2.5 mL filter sterilized ~~NH4Cl~~ (0.2 g/mL) or 0.5 g dry		2.5 mL filter sterilized NH<sub>4</sub>Cl (0.2 g/mL) or 0.5 g dry
	10 mL 20% d-glucose or 2 g dry		10 mL 20% d-glucose or 2 g dry

	Glass distilled & autoclaved ~~H2O~~ to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).		Glass distilled & autoclaved H<sub>2</sub>O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).

	Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/ml (or any antibiotics used to select the strains).		Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/ml (or any antibiotics used to select the strains).

	Grow 5mL overnight culture in same media to inoculate 500mL M9.		Grow 5mL overnight culture in same media to inoculate 500mL M9.
	Shake culture at 37 <sup>o</sup>C until an OD<sub>600</sub> of ~~07+/-~~0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).		Shake culture at 37 <sup>o</sup>C until an OD<sub>600</sub> of 0.7
			± 0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).

Bozhang at 03:40, 23 December 2012

2012-12-23T03:40:54Z

← Older revision		Revision as of 03:40, 23 December 2012
Line 2:		Line 2:
	[[Category:Protein expression]]		[[Category:Protein expression]]
	[[Category:Metabolomics]]		[[Category:Metabolomics]]
	~~1. General Protocols For Minimal Media Protein Expression~~

	Expression Protocol in M9 Minimal Media via T7 Promoter:The following protocol has been used successfully to ~~15N~~ or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:		1. General Protocols For Minimal Media Protein Expression
			Expression Protocol in M9 Minimal Media via T7 Promoter:The following protocol has been used successfully to {{SimpleNuclide2\|N\| 15}} or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:

	~~64g Na2HPO4~~-~~7H2O~~		64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O
	~~15g KH2PO4~~		15 g KH<sub>2</sub>PO<sub>4</sub>
	2.5g NaCl		2.5 g NaCl

	H2O to final 1L volume and autoclave		H2O to final 1L volume and autoclave
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	100mL of 5xM9 salts		100mL of 5xM9 salts
	1 mL ~~1M MgSO4~~		1 mL 1 M MgSO<sub>4</sub>
	50 uL ~~1M CaCl2~~		50 uL 1 M CaCl<sub>2</sub>
	5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)		5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
	2.5 mL filter sterilized NH4Cl (0.2 g/mL) or 0.5g dry		2.5 mL filter sterilized NH4Cl (0.2 g/mL) or 0.5 g dry
	10 mL 20% d-glucose or 2g dry		10 mL 20% d-glucose or 2 g dry

	Glass distilled & autoclaved H2O to final volume of ~~500mL~~ pH solution to 7.3 and filter sterilize (0.2u filter).		Glass distilled & autoclaved H2O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).

	Introduce media to a pre-autoclaved, wide-bottom (baffled) 2L flask and add ampicillin to a final concentration of 70-100 ug/ml (or any antibiotics used to select the strains).		Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/ml (or any antibiotics used to select the strains).

	Grow 5mL overnight culture in same media to inoculate 500mL M9.		Grow 5mL overnight culture in same media to inoculate 500mL M9.
	Shake culture at ~~37oC~~ until an ~~OD600~~ of 07+/-0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).		Shake culture at 37 <sup>o</sup>C until an OD<sub>600</sub> of 07+/-0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).


	2. General Protocols For LS Media		2. General Protocols For LS Media

	For 1L media preparation,		For 1 L media preparation,
	~~10g~~ Bacto-tryptone		10 g Bacto-tryptone
	5g yeast extract		5 g yeast extract
	~~10g~~ NaCl		10 g NaCl

	Sterilized by autoclave		Sterilized by autoclave

Bozhang at 00:10, 22 September 2012

2012-09-22T00:10:47Z

← Older revision		Revision as of 00:10, 22 September 2012
Line 1:		Line 1:
	[[Category:Protocols]]		[[Category:Protocols]]
	[[Category:Protein expression]]		[[Category:Protein expression]]
			[[Category:Metabolomics]]
	1. General Protocols For Minimal Media Protein Expression		1. General Protocols For Minimal Media Protein Expression

Wiki Administrator: 1 revision: Protocols

2012-03-13T00:02:21Z

1 revision: Protocols

← Older revision	Revision as of 00:02, 13 March 2012
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