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 PCR product was transformed by following the Transformation Protocol
  • The two solutions of 1μL of DPN1 was added to 45μL of K110A (f) and K110A (r) plasmid that had been stored in the freezer were used for cell transformation.
  • 1 vial of the cells was thawed on ice. 10ng of DNA in 5μL was added to 40 microliters of cells which were then heat shocked by incubating in a 42°C water bath for 30 seconds.
  • Upon removal, the vial was placed immediately on ice.
  • 200μL of SOC media was added to one solution of cells while 200 μL of LB was added to the other solution of cells. Both vials were shaken at 275 rpm for 1 hour.
  • Next, 25 μL of transformed cells were plated on one LB plate petridish (which was prewarmed and contained LB and kanamycin) while 200 μL of transformed cells was placed on the other. *The plates were incubated at 37°C overnight.
  • 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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