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

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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: 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 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.