Neidhardt EZ Rich Defined - Revision history

Smoore at 18:17, 3 July 2008

Smoore — Thu, 03 Jul 2008 18:17:43 GMT

←Older revision		Revision as of 18:17, 3 July 2008
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	**'''Teknova's response''': The M2001 used in your M2006 MOPS Minimal Kit contains temperature-sensitive components that require storage at -20°C. Storing this media for long term at 4°C will compromise the potency and may cause subcomponents to fall out of solution, just as you've experienced. I don't believe the particles you're observing in the solution are either fungus or mold, but rather iron sulfate that has precipitated out. To prevent this from occurring in the future, it would be advantageous to aliquot the mixed media, freeze, and thaw only the quantity you need. In my experience, even keeping the mixed solution refrigerated for more than a couple of days will severely reduce it's potency. Unfortunately, discarding the solution may be necessary.		**'''Teknova's response''': The M2001 used in your M2006 MOPS Minimal Kit contains temperature-sensitive components that require storage at -20°C. Storing this media for long term at 4°C will compromise the potency and may cause subcomponents to fall out of solution, just as you've experienced. I don't believe the particles you're observing in the solution are either fungus or mold, but rather iron sulfate that has precipitated out. To prevent this from occurring in the future, it would be advantageous to aliquot the mixed media, freeze, and thaw only the quantity you need. In my experience, even keeping the mixed solution refrigerated for more than a couple of days will severely reduce it's potency. Unfortunately, discarding the solution may be necessary.
	**Note that cells still seem to be able to grow in the media, though the integrity of media is likely compromised.		**Note that cells still seem to be able to grow in the media, though the integrity of media is likely compromised.
		+	*You might notice a significant lag if you start cultures from diluted overnights grown in LB. In the Niedhardt paper, there is data showing that large dilutions from log-phase cultures cause substantial lags (like hours), but these can be completely avoided by adding 10 mM NaCO<sub>3</sub>. I have found that making the RDM with 10% LB really helps get them going. Although this goes against the "defined" mantra, I haven't noticed an problem with reproducability when I do this. --[[User:Smoore\|Smoore]] 18:17, 3 July 2008 (UTC)

	==References==		==References==
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	[[Category:Material]] [[Category:Defined Media]]		[[Category:Material]] [[Category:Defined Media]]
		+	[[Category:Escherichia coli]]

Torsten Waldminghaus: add tags

Torsten Waldminghaus — Tue, 11 Mar 2008 15:39:07 GMT

add tags

←Older revision		Revision as of 15:39, 11 March 2008
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	==References==		==References==
	* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747		* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747
		+
		+
		+	[[Category:Material]] [[Category:Defined Media]]

Jason R. Kelly: /* Background */

Jason R. Kelly — Fri, 09 Feb 2007 23:48:50 GMT

Background

←Older revision		Revision as of 23:48, 9 February 2007
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	*Concentrations of the major nutrients can be varied independently (C, P, N, S)		*Concentrations of the major nutrients can be varied independently (C, P, N, S)
	*Concentrations of all constituents are defined		*Concentrations of all constituents are defined
-	*Growth rate should be similar to that of 'E. coli' in rich media such as LB	+	*Growth rate should be similar to that of ''E. coli'' in rich media such as LB
	*Supports a high density of cell growth		*Supports a high density of cell growth
	*Stable and gives reproducible results		*Stable and gives reproducible results

Barry Canton: /* Endy Lab Materials Sourcing & Usage */

Barry Canton — Thu, 30 Nov 2006 16:12:57 GMT

Endy Lab Materials Sourcing & Usage

←Older revision		Revision as of 16:12, 30 November 2006
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	==Endy Lab Materials Sourcing & Usage==		==Endy Lab Materials Sourcing & Usage==
	*We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from [http://www.teknova.com Teknova] because they are tedious to make. The Endy Lab has an account with [http://www.teknova.com Teknova] (see [[Barry Canton \| Barry]] for details).		*We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from [http://www.teknova.com Teknova] because they are tedious to make. The Endy Lab has an account with [http://www.teknova.com Teknova] (see [[Barry Canton \| Barry]] for details).
-	*The kitchen makes up batches of 10X MOPS Mixture for the us.	+	*The kitchen makes up batches of 10X MOPS Mixture for the us with some assistance. Here is the [[Endy:Neidhardt EZ Rich Defined\|recipe]] we use to make the 10X MOPS mixture.
	*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> for the us.		*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> for the us.
	*Currently, because some of the ingredients are stored in our freezer we are making up the final media mix. In the future we may get the kitchen to make up the whole thing except for the carbon source so people can choose what they want to use. As listed in the protocol section, the carbon source should be glycerol to conform to the working SBWG media standards.		*Currently, because some of the ingredients are stored in our freezer we are making up the final media mix. In the future we may get the kitchen to make up the whole thing except for the carbon source so people can choose what they want to use. As listed in the protocol section, the carbon source should be glycerol to conform to the working SBWG media standards.

Barry Canton: /* Protocol */

Barry Canton — Thu, 22 Jun 2006 21:07:57 GMT

Protocol

←Older revision		Revision as of 21:07, 22 June 2006
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	==Protocol==		==Protocol==
-	#The following protocol makes ~~1 L~~ of Media	+	#The following protocol makes 1L of Media
	#All of the ingredients have already been filter sterilized or autoclaved as appropriate.		#All of the ingredients have already been filter sterilized or autoclaved as appropriate.
	#To a 1 L flask add each of the following ingredients listed in the table below.		#To a 1 L flask add each of the following ingredients listed in the table below.

Reshma P. Shetty: /* Notes */

Reshma P. Shetty — Thu, 02 Mar 2006 21:44:27 GMT

Notes

←Older revision		Revision as of 21:44, 2 March 2006
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	==Notes==		==Notes==
	*Tricine is added to the MOPS mixture to chelate Fe ions to act as an Fe reservoir.		*Tricine is added to the MOPS mixture to chelate Fe ions to act as an Fe reservoir.
		+	*I had been storing made up media at 4°C and began to notice some brown precipitate at the bottom of the bottle. This precipitate would not go back into solution when the media was warmed to 37°C. I contacted Teknova about this.
		+	**'''Teknova's response''': The M2001 used in your M2006 MOPS Minimal Kit contains temperature-sensitive components that require storage at -20°C. Storing this media for long term at 4°C will compromise the potency and may cause subcomponents to fall out of solution, just as you've experienced. I don't believe the particles you're observing in the solution are either fungus or mold, but rather iron sulfate that has precipitated out. To prevent this from occurring in the future, it would be advantageous to aliquot the mixed media, freeze, and thaw only the quantity you need. In my experience, even keeping the mixed solution refrigerated for more than a couple of days will severely reduce it's potency. Unfortunately, discarding the solution may be necessary.
		+	**Note that cells still seem to be able to grow in the media, though the integrity of media is likely compromised.

	==References==		==References==
	* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747		* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747

Barry Canton: /* Endy Lab Materials Sourcing & Usage */

Barry Canton — Tue, 17 Jan 2006 21:16:12 GMT

Endy Lab Materials Sourcing & Usage

←Older revision		Revision as of 21:16, 17 January 2006
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	==Endy Lab Materials Sourcing & Usage==		==Endy Lab Materials Sourcing & Usage==
-	*We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from [http://www.teknova.com Teknova] because they are tedious to make. The Endy Lab has an account with [http://www.teknova.com Teknova] (see Barry for details).	+	*We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from [http://www.teknova.com Teknova] because they are tedious to make. The Endy Lab has an account with [http://www.teknova.com Teknova] (see [[Barry Canton \| Barry]] for details).
	*The kitchen makes up batches of 10X MOPS Mixture for the us.		*The kitchen makes up batches of 10X MOPS Mixture for the us.
	*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> for the us.		*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> for the us.

Barry Canton: /* Materials Sourcing & Usage */

Barry Canton — Wed, 04 Jan 2006 21:39:40 GMT

Materials Sourcing & Usage

←Older revision		Revision as of 21:39, 4 January 2006
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	*Due to the heat lability of the MOPS buffer, it cannot be autoclaved.		*Due to the heat lability of the MOPS buffer, it cannot be autoclaved.

-	==Materials Sourcing & Usage==	+	==Endy Lab Materials Sourcing & Usage==
	*We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from [http://www.teknova.com Teknova] because they are tedious to make. The Endy Lab has an account with [http://www.teknova.com Teknova] (see Barry for details).		*We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from [http://www.teknova.com Teknova] because they are tedious to make. The Endy Lab has an account with [http://www.teknova.com Teknova] (see Barry for details).
-	*The kitchen makes up batches of 10X MOPS Mixture for us.	+	*The kitchen makes up batches of 10X MOPS Mixture for the us.
-	*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> for us.	+	*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> for the us.
	*Currently, because some of the ingredients are stored in our freezer we are making up the final media mix. In the future we may get the kitchen to make up the whole thing except for the carbon source so people can choose what they want to use. As listed in the protocol section, the carbon source should be glycerol to conform to the working SBWG media standards.		*Currently, because some of the ingredients are stored in our freezer we are making up the final media mix. In the future we may get the kitchen to make up the whole thing except for the carbon source so people can choose what they want to use. As listed in the protocol section, the carbon source should be glycerol to conform to the working SBWG media standards.
	*I have 4 L of the final media made up so people can test their experiments in this media before switching over entirely. Please see me if you want to get some --[[User:Bcanton\|Bcanton]] 14:16, 7 Jun 2005 (EDT)		*I have 4 L of the final media made up so people can test their experiments in this media before switching over entirely. Please see me if you want to get some --[[User:Bcanton\|Bcanton]] 14:16, 7 Jun 2005 (EDT)

Barry Canton: /* Background */

Barry Canton — Wed, 04 Jan 2006 21:34:45 GMT

Background

←Older revision		Revision as of 21:34, 4 January 2006
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	*Low background fluorescence, can be used for fluorescent measurements		*Low background fluorescence, can be used for fluorescent measurements
	*Excellent pH buffering capacity		*Excellent pH buffering capacity
		+	*The levels of micronutrients (eg. copper, manganese etc.) are set to a level that is saturating for growth but also sufficiently low that there is no possibility of growth inhibition. Variance in the levels of these micronutrients due to contamination of glassware or water source should not move the concentrations out of this optimum range.
		+
		+	It also has the following disadvantages -
		+	*It is expensive if bought from a commercial supplier, and it is slow to make up on-site.
		+	*Some of the constituents and their levels are arbitrarily chosen. For example, NaCl is added to the medium, yet it function in aiding bacterial growth was unknown at the time of development.
		+	*Due to the heat lability of the MOPS buffer, it cannot be autoclaved.

	==Materials Sourcing & Usage==		==Materials Sourcing & Usage==

Barry Canton: /* Protocol */

Barry Canton — Wed, 04 Jan 2006 21:21:39 GMT

Protocol

←Older revision		Revision as of 21:21, 4 January 2006
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	\| TOTAL \|\| 1 L		\| TOTAL \|\| 1 L
	\|}		\|}
		+
		+	==Notes==
		+	*Tricine is added to the MOPS mixture to chelate Fe ions to act as an Fe reservoir.

	==References==		==References==
	* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747		* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747