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

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Objective

AuNP III

To synthesize gold nanoparticles (Au NP) 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 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.

Description

AuNP III

  1. Combine 10 mL of 50 mM acetate buffer (pH 5.46) with 1.1×10-3 g of .0015 mM bovine serum albumin (BSA) and 4.0×10-4 g of 0.25 mM chloroauric acid (HAuCl4) in a capped test tube at room temperature.
  2. Repeat with 10 mL of distilled water instead of acetate buffer.
  3. Take UV vis spectra (200 nm to 800 nm) of buffer, water, and both reaction mixtures.
  4. Place reaction mixtures in an oven at 80°C for 6 hr under thermostatic conditions.
  5. Continue to take UV vis spectra of reaction mixture every 30 minutes.
  6. 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 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.


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

Notes

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




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