User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/09/27

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Objective

1. To synthesize gold nanoparticles using the same procedure as 08/31/11 and 09/07/11.

2. To perform Protein Expression by beginning culture bacteria.

3. To transform DNA coding for mutant GFP that was mutated on 09/20/11 into Novablue cells.

Description

1. AuNP synthesis

  1. Combine 10 mL of 50 mM acetate buffer (pH 5.46) with 0.974 mL of 0.0015 mM BSA and 0.581 mL of 0.25 mM HAuCl4 in a capped test tube at room temperature.
  2. Take UV vis spectra (200 nm to 800 nm) of a sample of reaction mixture at 70°C every 30 min.
  3. Keep sample cuvette in UV-Vis instrument at 70°C between taking spectra.
  4. After 2.5 hr, remove from instrument and cool to room temperature; leave sitting overnight.


2. Protein Expression Starter Culture Media, Protein Expression

  1. Make expression culture media.
  2. Prepare LB in a 250 mL Erlenmeyer flask.
    1. 0.875 g of LB
    2. 35 mL of water
  3. Cover the flask with foil.
  4. Autoclave.
  5. Allow flask to cool.
  6. Add 35 μL of 1000X ampicillin.
  7. Inoculate with the bacteria you are culturing by scraping frozen bacteria/glycerol mixture and adding to flask.
  8. Place the flask in a shaker/incubator and culture overnight at 37°C and 200 rpm.


3. DNA Transformation

  1. Digest non-methylated DNA with 1 μL Dpnl.
  2. Make LB/agar plates with ampicillin.
  3. Place sterile tube and DNA in ice bucket for 15 min.
  4. Add 5 μL of DNA and 40μL of cells to the bottom of the tube.
  5. Incubate on ice for 30 min.
  6. Heat shock DNA/cells at 42°C for 30 s.
  7. Incubate on ice for 5 min.
  8. Add 250 μL of SOC media.
  9. Incubate in shaker at 37°C for 1 hr.
  10. Spread 100 μL of cells on LB/agar plate.
  11. Store plate (inverted) in 37°C oven overnight.

Data

  • No cells grew on our group plate, indicating that the transformation was unsuccessful. However, one group's plate grew cells.
  • Data for the nanoparticle synthesis is as follows:

This graph shows absorbance vs. time

Image:Absorbance_v._time.png

This graph shows absorbance vs. wavelength

Image:Absorbance_v._wavelength.png

This graph shows the zoomed in section of the absorbance vs. wavelength at 550 nm.

Image:550nm_zoom.png

Notes

The AuNP solution turned purple while in the oven, indicating that nanoparticles actually were formed.



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