User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/24

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Transformation, protein expression, lysozyme Uv-Vis

Goals

  • Transform PCR product into E.Coli Cells
  • Induce ADA protein expression
  • Run Uv-Vis of the lysozyme and AuNP solution

Procedure


The only changes that were made are as follows:

  1. Of the two cell solutions made, 200μL of LB media was added to one of the solutions while 200 μL of SOC media was added to the other solution of cells. The two solutions were then shaken at 275 rpm for 1 hr.
  2. 25 μL of transformed cells was plated on one LB Petri dish, while 200 μL of transformed cells was plated on the other. For K110A mutation, a total of 4 plates were made. 2 for K110A(f) and 1 for K110A (r).
  • ADA protein was expressed in the same manner as on 2012/09/19
    • This morning, Michael came in around 9am to centrifuge the starter culture media and resuspended the pellets in 4mL of fresh LB. These resuspended cells were then redivided into the expression culture media which was then inoculated in the shaker at 160rpm and 37°C. This was completed around 10am.
    • At approximately 1pm that same day, the IPTG was added into each of the flasks
    • 5mL of .4M IPTG solution was made and 1mL was placed into each of the 4 fernbach flasks.
    • .005L x.4M= .002 moles x 238.31g/mol= .477g IPTG in 5mL. Actual= .4754g in 5mL of water.
  • The 4 flasks were then shaken for 3 additional hours (until 4pm). After which the cells were spinned down in the centrifuge at 4500rpm for 15 minutes. Two rounds of centrifugation occurred because also the media could not fit into the centrifuge jars at once.
  • Supernatant was poured out and 30mL of binding buffer was used to resusped the cells. The flask of buffer and cells was then placed in a -80°C freezer for 1 week.
  • For information regarding the UV-Vis spectras of the AuNPs in Lysozyme please see either Mikes notebook or Marys notebook. The lysozyme did create purple gold nanoparticle fibers upon removal from the oven.




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