User:Catherine Koenigsknecht/Notebook/Experimental Biological Chemistry/2011/11/02

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Entry title

Objective

Use newly opened gold to make gold protein fibers to see if it affects the formation of the fibers. PCR product from yesterday needs to be transformed into E. coli.

Description

  1. Add 1 mL 15.5 uM BSA stock and 1 mL 2.5 mM HAuCl stock solutions to two test tubes. Add 8 mL distilled water to each test tube.
  2. Place test tubes in oven at 80C
  3. Remove one test tube every 30 min and let sit for 10 min. Keep the other test tube in the oven continuously.

PCR

  1. The wax was removed from the PCR product.
  2. 1μL of DpnI was added to the PCR product and it was put on a heat block at 37°C for one hour.
  3. 10μL of the PCR product was mixed with 2μL of 6x loading dye, and this was loaded into a 1.2% agarose gel.
  4. Take a sterile eppendorf tube and place it on ice.
  5. Mix 30μL of NovaBlue Competent E.coli with 5μL of the PCR product in the cold, sterile tube.
  6. Incubate this mixture for 30 minutes on ice.
  7. Transfer the tube to a heat block at 42°C for 30 seconds, and then transfer the tube back to ice for 5 minutes.
  8. Add 250μL of SOC media to the cells/PCR product.
  9. Shake the mixture at 250rpm at 37°C for one hour.
  10. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
    • The LB plate was made with 1.75g LB, 1.4g Agar, and 70mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 35mL of the mixture was poured into a sterile petri dish. Then, 35μL of 100mg/mL ampicillin was added to 35mL of the mixture that was remained. This was mixed and also poured into a sterile petri dish.
  11. Incubate the plate overnight (inverted) at 37°C.

Data

  • Both tubes had a clear solution with purple fibers

Notes

This area is for any observations or conclusions that you would like to note.


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