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

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==Procedure==
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==Objectives==
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# The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol|PCR Mutation]] was followed  
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*Mutate ADA via PCR mutation
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# The primers specifications are as follows:
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**Mutated ADA is intended for nucleation of AuNPs
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*Produce chemiluminescence by adding luminol to HRP assay
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==PCR Mutation==
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* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol|PCR Mutation]] was followed  
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** The specifications of the forward primer used are as follows:
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[[Image:Primer212.png]]
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**1mL molecular biology grade water was added to the primer.
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**The volume of primer solution needed to prepare 1mL of 100ng/uL solution was calculated
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***0.49mg/1000μL = 490ng/μL
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***100ng/uL*1000uL / 490ng/uL = 204.08μL
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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.
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**Two solutiosn were prepared with the following:
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***1uL forward primer
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***1uL reverse primer
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***1uL wild strand ADA
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***5uL 10x Pfu buffer
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***40.6uL molecular biology grade water
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***0.4Turbo DNA polymerase 25mM dNTPs
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***1uL Turbo DNA polymerase solution added immediately before placing on thermocycler
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**Thermocycler was programmed as following:
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***Heat for 2 minutes at 95<sup>o</sup>C
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***Repeat the following 30 times:
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****95<sup>o</sup>C for 30 seconds
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****-51<sup>o</sup>C for 30 seconds
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****72<sup>o</sup>C for 60 seconds
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***Maintain at 72<sup>o</sup>C
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***Slowly cool to 0<sup>o</sup>C
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**Solutions were stored in the freezer.
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==HRP Chemiluniscence Assay==
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*[[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.
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*The reaction solutions prepared contained .18mM iodophenol, .1mM H2O2 and 3mM luminol in 1mL quartz cuvettes.
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*2mM HRP added to the solution did not produce luminescence, nor did 10mM.
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*20mM produced a blue light that faded over the course of 3 minutes.
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__NOTOC__
__NOTOC__

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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:

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
      • 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


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