User:Michael F. Nagle/Notebook/Chem 571/2012/10/16 - Revision history

Michael F. Nagle at 07:45, 7 December 2012

Michael F. Nagle — Fri, 07 Dec 2012 07:45:04 GMT

←Older revision		Revision as of 07:45, 7 December 2012
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	<!-- ##### 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. ##### -->
		+	==Objectives==
		+	*Mutate ADA via PCR mutation
		+	**Mutated ADA is intended for nucleation of AuNPs
		+	*Produce chemiluminescence by adding luminol to HRP assay
	==PCR Mutation==		==PCR Mutation==
	* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed		* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed

Michael F. Nagle: /* HRP Chemiluniscence Assay */

Michael F. Nagle — Fri, 07 Dec 2012 07:41:05 GMT

HRP Chemiluniscence Assay

←Older revision		Revision as of 07:41, 7 December 2012
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	**Solutions were stored in the freezer.		**Solutions were stored in the freezer.
	==HRP Chemiluniscence Assay==		==HRP Chemiluniscence Assay==
-	*[[User:Mary_Mendoza/Notebook/~~CHEM_571_Experimental_Biological\|Mary Mendoza~~]] prepared 30mM luminol stock. Iodophenol, HRP and H<sub>2</sub>O<sub>2</sub> stock prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/02\|10/2]] were used.	+	*[[User:Mary_Mendoza/Notebook/CHEM_571_Experimental_Biological_Chemistry_I/2012/10/16]] prepared 30mM luminol stock. Iodophenol, HRP and H<sub>2</sub>O<sub>2</sub> stock prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/02\|10/2]] were used.
	*The reaction solutions prepared contained .18mM iodophenol, .1mM H2O2 and 3mM luminol in 1mL quartz cuvettes.		*The reaction solutions prepared contained .18mM iodophenol, .1mM H2O2 and 3mM luminol in 1mL quartz cuvettes.
	*2mM HRP added to the solution did not produce luminescence, nor did 10mM.		*2mM HRP added to the solution did not produce luminescence, nor did 10mM.

Michael F. Nagle at 07:40, 7 December 2012

Michael F. Nagle — Fri, 07 Dec 2012 07:40:22 GMT

←Older revision		Revision as of 07:40, 7 December 2012
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	<!-- ##### 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. ##### -->
-	==~~Procedure~~==	+	==PCR Mutation==
	* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed		* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed
	** The specifications of the forward primer used are as follows:		** The specifications of the forward primer used are as follows:
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	**100ng/uL1000uL / 490ng/uL = 204.08μL		**100ng/uL1000uL / 490ng/uL = 204.08μL
	**[[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/10/16\|Keyun Wang]] prepared 100ng/uL solution of the matching forward primer.		**[[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/10/16\|Keyun Wang]] prepared 100ng/uL solution of the matching forward primer.
-	**~~Solution was~~ prepared with the following:	+	**Two solutiosn were prepared with the following:
	***1uL forward primer		***1uL forward primer
	***1uL reverse primer		***1uL reverse primer
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	***5uL 10x Pfu buffer		***5uL 10x Pfu buffer
	***40.6uL molecular biology grade water		***40.6uL molecular biology grade water
-	***1uL Turbo DNA polymerase solution
	***0.4Turbo DNA polymerase 25mM dNTPs		***0.4Turbo DNA polymerase 25mM dNTPs
-		+	***1uL Turbo DNA polymerase solution added immediately before placing on thermocycler
		+	**Thermocycler was programmed as following:
		+	***Heat for 2 minutes at 95<sup>o</sup>C
		+	***Repeat the following 30 times:
		+	****95<sup>o</sup>C for 30 seconds
		+	****-51<sup>o</sup>C for 30 seconds
		+	****72<sup>o</sup>C for 60 seconds
		+	***Maintain at 72<sup>o</sup>C
		+	***Slowly cool to 0<sup>o</sup>C
		+	**Solutions were stored in the freezer.
		+	==HRP Chemiluniscence Assay==
		+	*[[User:Mary_Mendoza/Notebook/CHEM_571_Experimental_Biological\|Mary Mendoza]] prepared 30mM luminol stock. Iodophenol, HRP and H<sub>2</sub>O<sub>2</sub> stock prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/02\|10/2]] were used.
		+	*The reaction solutions prepared contained .18mM iodophenol, .1mM H2O2 and 3mM luminol in 1mL quartz cuvettes.
		+	*2mM HRP added to the solution did not produce luminescence, nor did 10mM.
		+	*20mM produced a blue light that faded over the course of 3 minutes.
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	\|}		\|}

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Michael F. Nagle at 06:46, 7 December 2012

Michael F. Nagle — Fri, 07 Dec 2012 06:46:00 GMT

←Older revision		Revision as of 06:46, 7 December 2012
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	**[[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/10/16\|Keyun Wang]] prepared 100ng/uL solution of the matching forward primer.		**[[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/10/16\|Keyun Wang]] prepared 100ng/uL solution of the matching forward primer.
	**Solution was prepared with the following:		**Solution was prepared with the following:
-	*1uL forward primer 1uL reverse primer 1uL wild strand ADA 5uL 10x Pfu buffer 40.6uL molecular biology grade water 1uL Turbo DNA polymerase solution **0.4Turbo DNA polymerase 25mM dNTPs	+	***1uL forward primer
		+	***1uL reverse primer
		+	***1uL wild strand ADA
		+	***5uL 10x Pfu buffer
		+	***40.6uL molecular biology grade water
		+	***1uL Turbo DNA polymerase solution
		+	***0.4Turbo DNA polymerase 25mM dNTPs

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Michael F. Nagle at 06:45, 7 December 2012

Michael F. Nagle — Fri, 07 Dec 2012 06:45:38 GMT

←Older revision		Revision as of 06:45, 7 December 2012
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	==Procedure==		==Procedure==
	* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed		* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed
-	** The specifications of the primer used are as follows:	+	** The specifications of the forward primer used are as follows:
	[[Image:Primer212.png]]		[[Image:Primer212.png]]
	**1mL molecular biology grade water was added to the primer.		**1mL molecular biology grade water was added to the primer.
	**The volume of primer solution needed to prepare 1mL of 100ng/uL solution was calculated		**The volume of primer solution needed to prepare 1mL of 100ng/uL solution was calculated
-	~~Target concentration: 100ng/uL~~
	***0.49mg/1000μL = 490ng/μL		***0.49mg/1000μL = 490ng/μL
	**100ng/uL1000uL / 490ng/uL = 204.08μL		**100ng/uL1000uL / 490ng/uL = 204.08μL
		+	**[[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/10/16\|Keyun Wang]] prepared 100ng/uL solution of the matching forward primer.
		+	**Solution was prepared with the following:
		+	*1uL forward primer 1uL reverse primer 1uL wild strand ADA 5uL 10x Pfu buffer 40.6uL molecular biology grade water 1uL Turbo DNA polymerase solution **0.4Turbo DNA polymerase 25mM dNTPs

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Michael F. Nagle at 06:38, 7 December 2012

Michael F. Nagle — Fri, 07 Dec 2012 06:38:40 GMT

←Older revision		Revision as of 06:38, 7 December 2012
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	==Procedure==		==Procedure==
-	# The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed	+	* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed
-	# The specifications of the primer used are as follows:	+	** The specifications of the primer used are as follows:
-		+	[[Image:Primer212.png]]
		+	**1mL molecular biology grade water was added to the primer.
		+	**The volume of primer solution needed to prepare 1mL of 100ng/uL solution was calculated
		+	Target concentration: 100ng/uL
		+	***0.49mg/1000μL = 490ng/μL
		+	**100ng/uL1000uL / 490ng/uL = 204.08μL

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Michael F. Nagle: /* Procedure */

Michael F. Nagle — Fri, 26 Oct 2012 12:00:05 GMT

Procedure

←Older revision		Revision as of 12:00, 26 October 2012
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	==Procedure==		==Procedure==
	# The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed		# The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed
-	# The ~~primers~~ specifications are as follows:	+	# The specifications of the primer used are as follows:
-		+

Michael F. Nagle at 11:59, 26 October 2012

Michael F. Nagle — Fri, 26 Oct 2012 11:59:29 GMT

←Older revision		Revision as of 11:59, 26 October 2012
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	<!-- ##### 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~~==	+	==Procedure==
-	* Insert content here...	+	# The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol\|PCR Mutation]] was followed
		+	# The primers specifications are as follows:
		+

Michael F. Nagle: Autocreate 2012/10/16 Entry for User:Michael_F._Nagle/Notebook/Chem_571

Michael F. Nagle — Wed, 24 Oct 2012 18:50:29 GMT

Autocreate 2012/10/16 Entry for User:Michael_F._Nagle/Notebook/Chem_571

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==Entry title==
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