- 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
- 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.