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

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:

• • 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 95^oC
    • Repeat the following 30 times:
      • 95^oC for 30 seconds
      • -51^oC for 30 seconds
      • 72^oC for 60 seconds
    • Maintain at 72^oC
    • Slowly cool to 0^oC
  • 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 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.