|
Objective
Determine the best way to make gold protein fibers. Introduce a mutation into GFP.
Description
Biomineralization of Gold Protein Fibers
- Mix four test tubes in the following way (add chemicals in the order written):
- Tube 1: 1mL 15.5μM BSA + 1mL 2.9mM HAuCl4 + 8mL 50mM Acetate buffer, pH 3.6
- Tube 2: 1mL 15.5μM BSA + 1mL 2.9mM HAuCl4 + 8mL 50mM Acetate buffer, pH 3.6
- Tube 3: 1mL 15.5μM BSA + 1mL 3M HCl + 8mL 50mM Acetate buffer, pH 3.6
- Tube 4: 1mL 15.5μM BSA + 1mL 3M HCl + 8mL 50mM Acetate buffer, pH 3.6
- Leave tubes 1&3 in the 80°C oven continuously over a 3 hour period.
- Put tubes 2&4 in the 80°C for a half hour, remove and store at room temperature for 10 minutes. Repeat this process 4 times.
- Store all of the test tubes at room temperature (wrapped in aluminum foil).
PCR
- Combine 5μL 10x Pfu buffer, 1μL DIC GFPcorr forward (5'-TACgacgatgacgataagtgtcgatggggatccgaattc-3'), 1μL GFPcorr reverse(5'-GAATTCGGATCCCCATCGACACTTATCGTCATCGTCGTA-3'), 1μL wild-type GFP DNA, 1μL dNTP mix, 40μL sterile H2O, and 1μL Pfu Turbo in a sterile PCR tube.
- Add 50μL of Bio Rad Chill Out™ liquid wax into the PCR tube.
- Place the PCR tube in the thermocycler:
- 30 sec at 95°C, 30 sec at 60°C, 7 min at 72°C
- Repeat those three steps 19 times
- 10 min at 72°C
- Overnight at 4°C
Data
- Tube 1 had a clump of black (with a purplish tint) fibers.
- Tube 2 had more purplish fibers that were not clumped as much.
- Tubes 3&4 remained clear.
|
AU Biomaterials Design Lab
