User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/24 - Revision history

Puja Mody: /* Procedure */

Puja Mody — Sat, 24 Nov 2012 19:05:42 GMT

Procedure

←Older revision		Revision as of 19:05, 24 November 2012
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	==Procedure==		==Procedure==
	* The PCR product was transformed by following the [[AU Biomaterials Design Lab:Protocols/Transformation Protocol\| Transformation Protocol]]		* The PCR product was transformed by following the [[AU Biomaterials Design Lab:Protocols/Transformation Protocol\| Transformation Protocol]]
-		+	*The two solutions of 1μL of DPN1 was added to 45μL of K110A (f) and K110A (r) plasmid that had been stored in the freezer were used for cell transformation.
-		+	* 1 vial of the cells was thawed on ice. 10ng of DNA in 5μL was added to 40 microliters of cells which were then heat shocked by incubating in a 42°C water bath for 30 seconds.
-	The ~~only changes~~ that were ~~made are as follows:~~	+	*Upon removal, the vial was placed immediately on ice.
-	~~# Of~~ the ~~two cell solutions made~~, 200μL of LB media was added to one of ~~the solutions~~ while 200 μL of ~~SOC media~~ was added to the other solution of cells. ~~The two solutions~~ were ~~then~~ shaken at 275 rpm for 1 hr.	+	*200μL of SOC media was added to one solution of cells while 200 μL of LB was added to the other solution of cells. Both vials were shaken at 275 rpm for 1 hour.
-	#25 μL of transformed cells ~~was~~ plated on one LB ~~Petri dish,~~ while 200 μL of transformed cells was ~~plated~~ on the other. ~~For K110A mutation, a total of 4~~ plates were ~~made. 2 for K110A(f) and 1 for K110A (r)~~.	+	Next, 25 μL of transformed cells were plated on one LB plate petridish (which was prewarmed and contained LB and kanamycin) while 200 μL of transformed cells was placed on the other. The plates were incubated at 37°C overnight.

	* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]		* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]

Puja Mody: /* Procedure */

Puja Mody — Sat, 24 Nov 2012 19:02:50 GMT

Procedure

←Older revision		Revision as of 19:02, 24 November 2012
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	*Supernatant was poured out and 30mL of binding buffer was used to resusped the cells. The flask of buffer and cells was then placed in a -80°C freezer for 1 week.		*Supernatant was poured out and 30mL of binding buffer was used to resusped the cells. The flask of buffer and cells was then placed in a -80°C freezer for 1 week.

-	* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/24\| Marys notebook]]. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.	+	* For information regarding the UV-Vis spectras of the AuNPs in Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/24\| Marys notebook]]. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.

Puja Mody: /* Procedure */

Puja Mody — Sat, 24 Nov 2012 18:58:10 GMT

Procedure

←Older revision		Revision as of 18:58, 24 November 2012
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	* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]		* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]
-	** This morning, Michael came in around 9am to centrifuge the starter culture media and resuspended the pellets in 4mL of fresh LB. These resuspended cells were then redivided into the expression culture media which was then inoculated in the shaker at 160rpm and 37°C. ~~Finally~~, the IPTG was added ~~at around 10am.~~	+	** This morning, Michael came in around 9am to centrifuge the starter culture media and resuspended the pellets in 4mL of fresh LB. These resuspended cells were then redivided into the expression culture media which was then inoculated in the shaker at 160rpm and 37°C. This was completed around 10am.
		+	** At approximately 1pm that same day, the IPTG was added into each of the flasks
	** 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.		** 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.
	** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.		** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.

-	* The 4 flasks were then shaken for 4 hours.	+	* The 4 flasks were then shaken for 3 additional hours (until 4pm). After which the cells were spinned down in the centrifuge at 4500rpm for 15 minutes. Two rounds of centrifugation occurred because also the media could not fit into the centrifuge jars at once.
		+	*Supernatant was poured out and 30mL of binding buffer was used to resusped the cells. The flask of buffer and cells was then placed in a -80°C freezer for 1 week.

	* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/24\| Marys notebook]]. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.		* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/24\| Marys notebook]]. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.

Puja Mody: /* Procedure */

Puja Mody — Thu, 22 Nov 2012 00:16:44 GMT

Procedure

←Older revision		Revision as of 00:16, 22 November 2012
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	* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]		* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]
		+	** This morning, Michael came in around 9am to centrifuge the starter culture media and resuspended the pellets in 4mL of fresh LB. These resuspended cells were then redivided into the expression culture media which was then inoculated in the shaker at 160rpm and 37°C. Finally, the IPTG was added at around 10am.
	** 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.		** 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.
	** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.		** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.
		+
		+	* The 4 flasks were then shaken for 4 hours.

	* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/24\| Marys notebook]]. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.		* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/24\| Marys notebook]]. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.

Puja Mody: /* Procedure */

Puja Mody — Thu, 22 Nov 2012 00:08:58 GMT

Procedure

←Older revision		Revision as of 00:08, 22 November 2012
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	==Procedure==		==Procedure==
	* The PCR product was transformed by following the [[AU Biomaterials Design Lab:Protocols/Transformation Protocol\| Transformation Protocol]]		* The PCR product was transformed by following the [[AU Biomaterials Design Lab:Protocols/Transformation Protocol\| Transformation Protocol]]
		+
		+
		+	The only changes that were made are as follows:
		+	# Of the two cell solutions made, 200μL of LB media was added to one of the solutions while 200 μL of SOC media was added to the other solution of cells. The two solutions were then shaken at 275 rpm for 1 hr.
		+	#25 μL of transformed cells was plated on one LB Petri dish, while 200 μL of transformed cells was plated on the other. For K110A mutation, a total of 4 plates were made. 2 for K110A(f) and 1 for K110A (r).
		+
	* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]		* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]
	** 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.		** 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.

Puja Mody: /* Procedure */

Puja Mody — Wed, 31 Oct 2012 16:50:41 GMT

Procedure

←Older revision		Revision as of 16:50, 31 October 2012
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	** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.		** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.

-	* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or ~~marys~~ notebook. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.	+	* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/24\| Marys notebook]]. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.

Puja Mody: /* Procedure */

Puja Mody — Fri, 26 Oct 2012 05:36:11 GMT

Procedure

←Older revision		Revision as of 05:36, 26 October 2012
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	** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.		** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.

-	* For information regarding the Lysozyme please see either ~~mikes~~ notebook or marys notebook. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.	+	* For information regarding the Lysozyme please see either [[User:Michael F. Nagle/Notebook/Chem 571/2012/10/24\| Mikes notebook]] or marys notebook. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.

Puja Mody: /* Procedure */

Puja Mody — Wed, 24 Oct 2012 19:20:47 GMT

Procedure

←Older revision		Revision as of 19:20, 24 October 2012
Line 19:		Line 19:
	** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.		** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.

-	* For information regarding the Lysozyme please see either mikes notebook or marys notebook.	+	* For information regarding the Lysozyme please see either mikes notebook or marys notebook. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.

Puja Mody at 17:21, 24 October 2012

Puja Mody — Wed, 24 Oct 2012 17:21:56 GMT

←Older revision		Revision as of 17:21, 24 October 2012
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	\| colspan="2"\|		\| colspan="2"\|
	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-	==~~Entry title~~==	+	==Transformation, protein expression, lysozyme Uv-Vis==
-	* ~~Insert content here~~...	+
		+	==Goals==
		+	*Transform PCR product into E.Coli Cells
		+	* Induce ADA protein expression
		+	*Run Uv-Vis of the lysozyme and AuNP solution
		+
		+	==Procedure==
		+	* The PCR product was transformed by following the [[AU Biomaterials Design Lab:Protocols/Transformation Protocol\| Transformation Protocol]]
		+	* ADA protein was expressed in the same manner as on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19\| 2012/09/19]]
		+	** 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.
		+	** .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.
		+
		+	* For information regarding the Lysozyme please see either mikes notebook or marys notebook.
		+
		+

Puja Mody: Autocreate 2012/10/24 Entry for User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles

Puja Mody — Wed, 24 Oct 2012 17:15:23 GMT

Autocreate 2012/10/24 Entry for User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles

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