Difference between revisions of "User:Michael F. Nagle/Notebook/Chem 571/2012/10/16"

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==Procedure==
+
==Objectives==
 +
*Mutate ADA via PCR mutation
 +
**Mutated ADA is intended for nucleation of AuNPs
 +
*Produce chemiluminescence by adding luminol to HRP assay
 +
==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 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/uL*1000uL / 490ng/uL = 204.08μL
 
***100ng/uL*1000uL / 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.
 +
**Two solutiosn were prepared with the following:
 +
***1uL forward primer
 +
***1uL reverse primer
 +
***1uL wild strand ADA
 +
***5uL 10x Pfu buffer
 +
***40.6uL molecular biology grade water
 +
***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_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.
 +
*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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__NOTOC__
 
__NOTOC__

Latest revision as of 00:45, 7 December 2012

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Objectives

  • Mutate ADA via PCR mutation
    • Mutated ADA is intended for nucleation of AuNPs
  • Produce chemiluminescence by adding luminol to HRP assay

PCR Mutation

  • The procedure for PCR Mutation was followed
    • The specifications of the forward primer used are as follows:

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
      • 0.49mg/1000μL = 490ng/μL
      • 100ng/uL*1000uL / 490ng/uL = 204.08μL
    • Keyun Wang prepared 100ng/uL solution of the matching forward primer.
    • Two solutiosn were prepared with the following:
      • 1uL forward primer
      • 1uL reverse primer
      • 1uL wild strand ADA
      • 5uL 10x Pfu buffer
      • 40.6uL molecular biology grade water
      • 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 95oC
      • Repeat the following 30 times:
        • 95oC for 30 seconds
        • -51oC for 30 seconds
        • 72oC for 60 seconds
      • Maintain at 72oC
      • Slowly cool to 0oC
    • Solutions were stored in the freezer.

HRP Chemiluniscence Assay