User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/27

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AuNP III, Expression, DNA Transformation Main project page
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To synthesize AuNP using the procedure developed here:

Bakshi, Mandeep S.; Kaur, Harpreet; Khullar, Poonam, Banipal, Tarlok, S.; Kaur, Gurinder; Singh, Narpinder Journal of Physical Chemistry C 2011, 115(7), 2982-2992

In addition, to repeat the experiment performed on 08/31/11 and 09/07/11.

Protein Expression

To begin culturing bacteria to express inteins.

DNA Transformation

To transform DNA coding for mutant GFP (mutated on 09/20/11) into Novablue cells.



  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. Place reaction mixture in an oven at 80°C for 2.5 hr under thermostatic conditions.
  4. After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.

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 1000× 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.

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 40 μL 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.



The absorbance values for the AuNP solution were subtracted from the absorbance values for a blank acetate buffer to create a set of corrected absorbance values. The corrected absorbance values at 550 nm were graphed against time. The graph shows a positive linear relationship, indicating that AuNPs were formed in solution.

In addition, a second graph showing corrected absorbance against wavelength was created, to show the absorbance levels across all of the wavelengths measured by the UV-vis.

A magnified version of this graph shows that absorbance values at around 550 nm increased over time, indicating the creation of AuNPs.

DNA Transformation

Plate did not appear to be successful; no cells grew.


The AuNP solution turned from clear to purple while in the oven, indicating that AuNPs were actually formed.

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