User:Laura Flynn/Notebook/Experimental Biological Chemistry/2011/09/27

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Objective

Three objectives were part of today's experiment. First, AuNP nanoparticle synthesis was repeated with constant heat. Second, bacteria was cultured to express inteins via protein expression. Lastly, DNA coding for mutant GFP (mutated on 09/20/11) was transformed.

Procedure

AUNP synthesis:

Protocol from [1], was followed in the same manner with one exception: Each cuvette was kept internally in the UV Vis at around 70 degrees Celsius.

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.

Results

Acetate as a Buffer:



As shown above, the absorbance increased as time increased.

The positive linear relationship suggests that AUNP were formed in the solution as time increased.

Notes

DNA transformation did not appear successful, as no cells grew.


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