User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19 - Revision history

Puja Mody: /* Procedure */

2012-11-24T19:01:46Z

Procedure

←Older revision		Revision as of 19:01, 24 November 2012
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	# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.		# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.
	--[[User:Abigail E. Miller\|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)it is not clear that this was done in the morning.		--[[User:Abigail E. Miller\|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)it is not clear that this was done in the morning.
-	~~# Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.~~	+	That afternoon:
-	# ~~Add~~ 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG\|IPTG]] to each flask in order to induce protein expression	+	# 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG\|IPTG]] was added to each flask in order to induce protein expression. The 4 fernbach flasks were then allowed to continue to shake for 3 more hours.
-	~~# Continue shaking~~ for 3-4 hours	+	#The cells were harvested by centrifuging at 4500rpm for 15 minutes. 2 runs of this had to be done because not all the media was able to fit in the centrifuge jars in one run.
-	# ~~Add 30 mL of the binding buffer into each flask~~	+	# The cells were then resuspended using 30mL of binding buffer for flasks 2-4 and 10mL separate for flask 1.
-	~~# Harvest the~~ cells by centrifuging at 4500rpm for 15 minutes	+	# These flasks were then placed in a -80°C freezer overnight.
-	~~# collect the cells and place in -80°C freezer~~	+
-		+
-	~~--[[User:Abigail E~~. ~~Miller\|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)~~this ~~is too much like~~ the ~~protocol. what did you actually do? at what time~~ was the ~~IPTG added? you did not measure the OD at 600nm~~. ~~what time did you harvest the~~ cells~~? therefore how long~~ were ~~they allowed to express? how many times did the cells have to be spun down? what deviations did you make from the protocol~~ - ~~I know you separated~~ flask 1 ~~form the other three~~. ~~that is not clear here~~.	+

	==Calculations==		==Calculations==

Abigail E. Miller: /* Calculations */

2012-10-07T14:54:28Z

Calculations

←Older revision		Revision as of 14:54, 7 October 2012
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	* Refer to [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/19\| Mary's Notebook]] for the actual amounts of buffer components measured out and added to solution.		* Refer to [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/19\| Mary's Notebook]] for the actual amounts of buffer components measured out and added to solution.
		+
		+	--[[User:Abigail E. Miller\|Abigail E. Miller]] 10:54, 7 October 2012 (EDT)did you have to adjust the pH?

	==Conclusions==		==Conclusions==

Abigail E. Miller: /* Procedure */

2012-10-07T14:53:14Z

Procedure

←Older revision		Revision as of 14:53, 7 October 2012
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	#This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB. For more details on the process in the morning, see [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/19\| Mary's Notebook]]		#This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB. For more details on the process in the morning, see [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/19\| Mary's Notebook]]
	# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.		# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.
		+	--[[User:Abigail E. Miller\|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)it is not clear that this was done in the morning.
	# Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.		# Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.
	# Add 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG\|IPTG]] to each flask in order to induce protein expression		# Add 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG\|IPTG]] to each flask in order to induce protein expression
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	# Harvest the cells by centrifuging at 4500rpm for 15 minutes		# Harvest the cells by centrifuging at 4500rpm for 15 minutes
	# collect the cells and place in -80°C freezer		# collect the cells and place in -80°C freezer
		+
		+	--[[User:Abigail E. Miller\|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)this is too much like the protocol. what did you actually do? at what time was the IPTG added? you did not measure the OD at 600nm. what time did you harvest the cells? therefore how long were they allowed to express? how many times did the cells have to be spun down? what deviations did you make from the protocol - I know you separated flask 1 form the other three. that is not clear here.

	==Calculations==		==Calculations==

Puja Mody: /* Procedure */

2012-10-03T06:42:35Z

Procedure

←Older revision		Revision as of 06:42, 3 October 2012
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	==Procedure==		==Procedure==
	*Protein Expression		*Protein Expression
-	#This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB.	+	#This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB. For more details on the process in the morning, see [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/19\| Mary's Notebook]]
	# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.		# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.
	# Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.		# Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.

Puja Mody: /* Conclusions */

2012-10-03T06:41:34Z

Conclusions

←Older revision		Revision as of 06:41, 3 October 2012
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	==Conclusions==		==Conclusions==
-	*sample 1 looked a little different when the samples were looked at this morning prior to centrifuging. It was a different shade of yellow in the erlenmeyer flask.	+	*sample 1 looked a little different when the samples were looked at this morning prior to centrifuging. It was a different shade of yellow in the erlenmeyer flask. As a result, for the rest of the protein expression, it will be separate from the other 3 made samples just in case there was anything amiss in that sample (sample 1).

Puja Mody: /* Goals */

2012-10-03T06:40:15Z

Goals

←Older revision		Revision as of 06:40, 3 October 2012
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	==Goals==		==Goals==
-	*Induce Protein expression.	+	*Induce [[AU Biomaterials Design Lab:Protocols/Protein Expression\| Protein expression]]
	* make the binding buffer and the elution buffer		* make the binding buffer and the elution buffer
	*Harvest the cells that were grown.		*Harvest the cells that were grown.

Puja Mody: /* Calculations */

2012-10-03T06:38:11Z

Calculations

←Older revision		Revision as of 06:38, 3 October 2012
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	# .5M NaCl= 14.609 g NaCl		# .5M NaCl= 14.609 g NaCl
	#.5 M imidazole= 17.019 g imidazole.		#.5 M imidazole= 17.019 g imidazole.
		+
		+	* Refer to [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/19\| Mary's Notebook]] for the actual amounts of buffer components measured out and added to solution.

	==Conclusions==		==Conclusions==

Puja Mody: /* Goals */

2012-09-26T08:43:58Z

Goals

←Older revision		Revision as of 08:43, 26 September 2012
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	*Induce Protein expression.		*Induce Protein expression.
	* make the binding buffer and the elution buffer		* make the binding buffer and the elution buffer
-	*Continue running AuNP tests-	+	*Harvest the cells that were grown.
-	*Harvest the cells that were grown.	+

	==Procedure==		==Procedure==

Puja Mody: /* Procedure */

2012-09-26T08:43:34Z

Procedure

←Older revision		Revision as of 08:43, 26 September 2012
Line 19:		Line 19:
	#This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB.		#This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB.
	# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.		# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media\|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.
-	# Incubate the expression cultures at ~~37C~~ and ~~160rpm~~ until the absorbance of the media at 600nm = 0.6.	+	# Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.
	# Add 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG\|IPTG]] to each flask in order to induce protein expression		# Add 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG\|IPTG]] to each flask in order to induce protein expression
	# Continue shaking for 3-4 hours		# Continue shaking for 3-4 hours
		+	# Add 30 mL of the binding buffer into each flask
	# Harvest the cells by centrifuging at 4500rpm for 15 minutes		# Harvest the cells by centrifuging at 4500rpm for 15 minutes
		+	# collect the cells and place in -80°C freezer

-	* Binding Buffer: 1 liter total solution at pH 7.4	+	==Calculations==

-	Theoretical calculations	+	* Binding Buffer: 1 liter total solution at pH 7.4.
		+	a. Theoretical calculations
	#20mM Tris= .020M/L Tris x1 L= .020 mols x 121.14 g/mol = 2.4228 g Tris.		#20mM Tris= .020M/L Tris x1 L= .020 mols x 121.14 g/mol = 2.4228 g Tris.
	** similar calculations were done for all compounds added into the buffer solution.		** similar calculations were done for all compounds added into the buffer solution.
	# .5M NaCl= 29.2195 g NaCl		# .5M NaCl= 29.2195 g NaCl
-	# 30mM Imidazole	+	# 30mM Imidazole= 2.0424 g Imidazole
		+
		+	* Elution Buffer: 500 mL total solution at pH 7.4
		+	a. Theoretical calculations
		+	#20mM Tris= .020 × .500= .01 mols × 121.14/ 1mol= 1.2114 g Tris
		+	** similar calculations/stoichiometry was done for all compounded added into the buffer solution.
		+	# .5M NaCl= 14.609 g NaCl
		+	#.5 M imidazole= 17.019 g imidazole.

	==Conclusions==		==Conclusions==

Puja Mody: /* Procedure */

2012-09-19T19:31:56Z

Procedure

←Older revision		Revision as of 19:31, 19 September 2012
Line 24:		Line 24:
	# Harvest the cells by centrifuging at 4500rpm for 15 minutes		# Harvest the cells by centrifuging at 4500rpm for 15 minutes

		+	* Binding Buffer: 1 liter total solution at pH 7.4

		+	Theoretical calculations
		+	#20mM Tris= .020M/L Tris x1 L= .020 mols x 121.14 g/mol = 2.4228 g Tris.
		+	** similar calculations were done for all compounds added into the buffer solution.
		+	# .5M NaCl= 29.2195 g NaCl
		+	# 30mM Imidazole

	==Conclusions==		==Conclusions==