User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/28

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AuNP IV, Expression II Main project page
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Objective

AuNP IV

To synthesize AuNPs 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

http://pubs.acs.org/doi/abs/10.1021/jp110296y


In addition, to repeat the experiment performed on 08/31/11, 09/07/11, and 09/27/11.


Protein Expression II

To continue the experiment on 09/27/11 involving culturing bacteria to express inteins.

Description

AuNP IV

  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 II

  1. Centrifuge the starter cultures at 4500 rpm for 15 min
  2. Re-suspend the cell pellets in 4mL of fresh LB.
  3. Inoculate the expression culture media by dividing the resuspended cells among the Fernbach flasks.
  4. Incubate the expression cultures at 37C and 160rpm until the absorbance of the media at 600 nm = 0.6.
  5. Add 1 mL of 0.4 M IPTG to each flask in order to induce protein expression
  6. Continue shaking for 3-4 hr
  7. Harvest the cells by centrifuging at 4500 rpm for 15 min at 4°C with a SH-3000(BK) rotor.
  8. Collect the cells in tris buffer (pH 9) and place in the -80°C freezer

Data

AuNP IV

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 after 60 min, 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. Absorbance data collected at 150 min from 200 nm-300 nm was ignored, because of outside interference with the UV-vis instrument.

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

Notes

At 150 min from 200 nm-300 nm, there was interference with the UV-vis, so that data was disregarded.




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