User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2011/11/01

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Objective

Determine the best way to make gold protein fibers. Introduce a mutation into GFP.

Description

Biomineralization of Gold Protein Fibers

  1. 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
  2. Leave tubes 1&3 in the 80°C oven continuously over a 3 hour period.
  3. 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.
  4. Store all of the test tubes at room temperature (wrapped in aluminum foil).

PCR

  1. 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.
  2. Add 50μL of Bio Rad Chill Out™ liquid wax into the PCR tube.
  3. 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.



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