User:Michael F. Nagle/Notebook/Chem 571/2012/10/24

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ADA expression

  • I came in several hours earlier than usual, at 9am, to resuspend cells in fresh LB.
  • Cells were centrifuged at 4500rpm for 15 minutes.
  • Supernatant was dumped and 4mL fresh LB was added to one tube, which was shaken to resuspend cells before the solution was transferred to the next one, which was also shaken. This was repeated until all cells were resuspended.
  • Cells were incubated at 160rpm and 37°C until 4pm.
  • Cells were again centrifuged at 4500rpm for 15 minutes. Supernatant was dumped and cell swere resuspended in binding buffer prepared by Puja Mody.
    • Cells were stored at -80oC

Transformation of Mutated Plasmid

  • Transformation protocol was followed, with the following modifications.
  • 1μL DPN1 was added to each plasmid solution.
  • A vial of E. Coli was thawed on ice.
  • 10ng DNA in 5μL solution was added to 40μL cells.
  • The cells were incubated in a 42oC water bath.
  • After 30 seconds, they were removed and placed on ice.
  • 200μL SOC media was added to one solution and 200μL LB was added to the other.
  • Both were incubated at 275rpm at room temperature for an hour.
  • 25μL cells were added to one petri dish and 200μL to another.
  • Both dishes contained LB and kanamycin.
  • The plates were incubated overnight at 37oC

UV/Vis of Au and BSA/Lysozyme

  1. Solutions with Lysozyme and BSA prepared [[last week were analyzed via UV/Vis. There was no peak at 520nm, so it was determined that heating at 50°C was insufficient. The solutions went back in the oven for 4 hours at 80°C, which is the same temperature BSA was seen succesfully unfolding at.
  2. Solutions with Au and BSA prepared last week were analyzed via UV/Vis.


Bsauvlast.png Lyzfirst.png