User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/18 - Revision history

Puja Mody: /* Procedure */

Puja Mody — Tue, 27 Nov 2012 21:33:24 GMT

Procedure

←Older revision		Revision as of 21:33, 27 November 2012
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	==Procedure==		==Procedure==
-	~~--[[User:Abigail E. Miller\|Abigail E. Miller]] 10:48, 7 October 2012 (EDT): note what hte starter and expression cultures are for; complete sentences...~~Following the protocol ~~xxxx~~, we created LB broth for growing cells....	+	*Following the Starter Culture media protocol and the expression culture media protocol, we created LB broth for growing cells. The Starter culture media was used mainly today and was eventually combined with the expression culture media.

-	* [[~~http://openwetware.org/wiki/~~AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media\| Started Culture Protocol]]	+	* [[AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media\| Started Culture Protocol]]
	** add 35 mL H<sub>2</sub>Oto each to 4 250mL erlenmeyer flask		** add 35 mL H<sub>2</sub>Oto each to 4 250mL erlenmeyer flask
	** [[AU_Biomaterials_Design_Lab:Materials/LB\|LB]] was added to each flask.		** [[AU_Biomaterials_Design_Lab:Materials/LB\|LB]] was added to each flask.
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	(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)		(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)

-	* [[~~http://openwetware.org/wiki/~~AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\| Expression Culture Media]]	+	* [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\| Expression Culture Media]]
	** 1L of distilled water were added to 4.8L Fernbach flasks		** 1L of distilled water were added to 4.8L Fernbach flasks
	**LB was added to each flask		**LB was added to each flask

Abigail E. Miller: /* Procedure */

Abigail E. Miller — Sun, 07 Oct 2012 14:48:18 GMT

Procedure

←Older revision		Revision as of 14:48, 7 October 2012
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	==Procedure==		==Procedure==
		+	--[[User:Abigail E. Miller\|Abigail E. Miller]] 10:48, 7 October 2012 (EDT): note what hte starter and expression cultures are for; complete sentences...Following the protocol xxxx, we created LB broth for growing cells....
		+
	* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media\| Started Culture Protocol]]		* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media\| Started Culture Protocol]]
	** add 35 mL H<sub>2</sub>Oto each to 4 250mL erlenmeyer flask		** add 35 mL H<sub>2</sub>Oto each to 4 250mL erlenmeyer flask

Puja Mody: /* Procedure */

Puja Mody — Wed, 03 Oct 2012 06:34:45 GMT

Procedure

←Older revision		Revision as of 06:34, 3 October 2012
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	* in actuality, 3.080 grams of tris was added.		* in actuality, 3.080 grams of tris was added.

		+
		+	Calculations for amount of water and tris needed to achieve desired concentrations:
		+
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Concentration of Tris (mM)'''
		+	\| align="center" style="background:#f0f0f0;"\|'''mL of water'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Amount of tris to be added (mL)'''
		+	\|-
		+	\| 0.05\|\|0.994\|\|0.00625
		+	\|-
		+	\| 5\|\|0.998\|\|0.0125
		+	\|-
		+	\| 5\|\|0.975\|\|0.025
		+	\|-
		+	\| 50\|\|0.95\|\|0.05
		+	\|-
		+	\| 100\|\|0.9\|\|0.1
		+	\|-
		+	\| 200\|\|0.8\|\|0.2
		+	\|-
		+	\| 500\|\|0.5\|\|0.5
		+	\|-
		+	\| 0.1\|\|0\|\|1
		+	\|}
		+
		+	(The tris and water were not combined with the AuNP/BSa solutions today)

Puja Mody: /* Procedure */

Puja Mody — Wed, 03 Oct 2012 06:31:39 GMT

Procedure

←Older revision		Revision as of 06:31, 3 October 2012
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	* Aseptic techniques were applied by executing the inoculation process near the proximity of a Bunsen burner. The aluminum foil tops of the Erlenmeyer containing the broths were flamed in between uncapping and capping. The metal tweezers were flamed before and after use. Sterile wood sticks were used to capture a colony of the ''E.Coli''		* Aseptic techniques were applied by executing the inoculation process near the proximity of a Bunsen burner. The aluminum foil tops of the Erlenmeyer containing the broths were flamed in between uncapping and capping. The metal tweezers were flamed before and after use. Sterile wood sticks were used to capture a colony of the ''E.Coli''
		+
		+	*The starter culture media flasks were then placed in a shaker incubator at 37°C at 237rpm overnight.
		+
		+	Preparation of Tris Buffer
		+	*pH 10- this buffer would be used with the AuNP/BSA made at a concentration of 70 [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/12\| last week]]
		+
		+	the desired concentration of the stock was .1 M tris and a total of 25mL of stock was made.
		+
		+	1 mol/ 1L x .025 L= .025 mol
		+
		+	.025 mol x 121.14 grams/1 mol = 3.0285 grams of tris in 25mL of water
		+	* in actuality, 3.080 grams of tris was added.
		+
		+

Puja Mody: /* Procedure */

Puja Mody — Wed, 03 Oct 2012 06:15:26 GMT

Procedure

←Older revision		Revision as of 06:15, 3 October 2012
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	* The bacteria was inoculated by retrieving a single colony from the petri dish and dropping it into the flask.		* The bacteria was inoculated by retrieving a single colony from the petri dish and dropping it into the flask.
-	**After all the broths ~~had~~ cooled down,	+	**After all the starter culture media broths cooled down, they were inoculate with transformed ''Escherichia coli''. Aseptic techniques are implemented to prevent any undesirable microbes from being included in the inoculation of the media.
		+	*''E. coli'' were transformed by the insertion of plasmid encoding for Adenosine Deaminase (ADA).
		+
		+	* Aseptic techniques were applied by executing the inoculation process near the proximity of a Bunsen burner. The aluminum foil tops of the Erlenmeyer containing the broths were flamed in between uncapping and capping. The metal tweezers were flamed before and after use. Sterile wood sticks were used to capture a colony of the ''E.Coli''
		+
		+
		+

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Puja Mody: /* Procedure */

Puja Mody — Wed, 03 Oct 2012 06:10:25 GMT

Procedure

←Older revision		Revision as of 06:10, 3 October 2012
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-	* Next, two of the erlenmeyer flasks and two of the Fernbach flasks were placed in the [[AU_Biomaterials_Design_Lab:Protocols/Autoclave\|Autoclave]] at a temperature of 121°C for 60minutes followed by a 30 minute cool down period. After ~~which,~~ the ~~other~~ four samples were placed in the autoclave for 50 minutes at the same temperature and were cooled down for 30 minutes as well. The autoclave is used in order to kill all other living organisms so that they do not react with the LB because we want to only grow/culture the E.Coli cells.	+	* Next, two of the erlenmeyer flasks and two of the Fernbach flasks were capped with aluminum foil and placed in the [[AU_Biomaterials_Design_Lab:Protocols/Autoclave\|Autoclave]] at a temperature of 121°C for 60minutes followed by a 30 minute cool down period. The autoclave is necessary in order to be sterilized and to ensure that no microbes from the surrounding environment could compete with the bacteria. After the first four samples were removed from the autoclave, the remaining 4 were placed in the autoclave for 50 minutes at the same temperature and were cooled down for 30 minutes as well. The samples had to be autoclaved separately because of lack of room in the autoclave. The autoclave is used in order to kill all other living organisms so that they do not react with the LB because we want to only grow/culture the E.Coli cells.

-	* ~~The~~ antibiotic Kanamycin ~~was~~ added ~~into each of the flasks~~. The stock solution of the antibiotic was 50mg/mL. The desired concentration of kanamycin for the flasks was 50μg/mL.	+	* While autoclaving the samples, the amount of antibiotic Kanamycin that would be added in was calculated. The stock solution of the antibiotic was 50mg/mL. The desired concentration of kanamycin for the flasks was 50μg/mL.
	Using the formula M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub>		Using the formula M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub>
	The volume or amount of kanamycin that would need to go into each of the flasks was 50 μg/mL x 35mL ÷50,000 μg		The volume or amount of kanamycin that would need to go into each of the flasks was 50 μg/mL x 35mL ÷50,000 μg
-	= .~~35mL~~ Kanamycin.	+	= .035mL Kanamycin.
-		+
		+	* The bacteria was inoculated by retrieving a single colony from the petri dish and dropping it into the flask.
		+	**After all the broths had cooled down,

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Puja Mody: /* Procedure */

Puja Mody — Wed, 03 Oct 2012 05:53:21 GMT

Procedure

←Older revision		Revision as of 05:53, 3 October 2012
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	(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)		(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)

-	* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|Expression Culture ~~Protocols~~]]	+	* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\| Expression Culture Media]]
	** 1L of distilled water were added to 4.8L Fernbach flasks		** 1L of distilled water were added to 4.8L Fernbach flasks
	**LB was added to each flask		**LB was added to each flask

Puja Mody: /* Procedure */

Puja Mody — Wed, 03 Oct 2012 05:50:43 GMT

Procedure

←Older revision		Revision as of 05:50, 3 October 2012
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	==Procedure==		==Procedure==
	* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media\| Started Culture Protocol]]		* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media\| Started Culture Protocol]]
-	** add 35 mL to each to 4 250mL erlenmeyer flask	+	** add 35 mL H<sub>2</sub>Oto each to 4 250mL erlenmeyer flask
	** [[AU_Biomaterials_Design_Lab:Materials/LB\|LB]] was added to each flask.		** [[AU_Biomaterials_Design_Lab:Materials/LB\|LB]] was added to each flask.
	*** Flask 1=.8793g		*** Flask 1=.8793g

Puja Mody: /* Procedure */

Puja Mody — Wed, 03 Oct 2012 05:49:38 GMT

Procedure

←Older revision		Revision as of 05:49, 3 October 2012
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	==Procedure==		==Procedure==
-	* ~~Starter Culture Protocol:~~ http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media	+	* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media\| Started Culture Protocol]]
	** add 35 mL to each to 4 250mL erlenmeyer flask		** add 35 mL to each to 4 250mL erlenmeyer flask
	** [[AU_Biomaterials_Design_Lab:Materials/LB\|LB]] was added to each flask.		** [[AU_Biomaterials_Design_Lab:Materials/LB\|LB]] was added to each flask.
Line 24:		Line 24:
	(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)		(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)

-	* ~~Expression culture Protocols:~~ http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media	+	* [[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|Expression Culture Protocols]]
	** 1L of distilled water were added to 4.8L Fernbach flasks		** 1L of distilled water were added to 4.8L Fernbach flasks
	**LB was added to each flask		**LB was added to each flask

Puja Mody: /* Procedure */

Puja Mody — Wed, 26 Sep 2012 08:34:02 GMT

Procedure

←Older revision		Revision as of 08:34, 26 September 2012
Line 38:		Line 38:
	* Next, two of the erlenmeyer flasks and two of the Fernbach flasks were placed in the [[AU_Biomaterials_Design_Lab:Protocols/Autoclave\|Autoclave]] at a temperature of 121°C for 60minutes followed by a 30 minute cool down period. After which, the other four samples were placed in the autoclave for 50 minutes at the same temperature and were cooled down for 30 minutes as well. The autoclave is used in order to kill all other living organisms so that they do not react with the LB because we want to only grow/culture the E.Coli cells.		* Next, two of the erlenmeyer flasks and two of the Fernbach flasks were placed in the [[AU_Biomaterials_Design_Lab:Protocols/Autoclave\|Autoclave]] at a temperature of 121°C for 60minutes followed by a 30 minute cool down period. After which, the other four samples were placed in the autoclave for 50 minutes at the same temperature and were cooled down for 30 minutes as well. The autoclave is used in order to kill all other living organisms so that they do not react with the LB because we want to only grow/culture the E.Coli cells.

-	* The antibiotic Kanamycin was added into each of the flasks.	+	* The antibiotic Kanamycin was added into each of the flasks. The stock solution of the antibiotic was 50mg/mL. The desired concentration of kanamycin for the flasks was 50μg/mL.
-		+	Using the formula M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub>
		+	The volume or amount of kanamycin that would need to go into each of the flasks was 50 μg/mL x 35mL ÷50,000 μg
		+	= .35mL Kanamycin.
		+

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