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

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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

  • Mary Mendoza prepared 30mM luminol stock. Iodophenol, HRP and H2O2 stock prepared 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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