User:Daniel Goodman/Notebook/Cluzel/2010/04/05

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Cluzel Lab Notebook
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Prepping Plasmid for Electroporation

PZE-12-CFP vector from JM Kim

Electroporations

ng/ul 260/280 260/230
56.3 ng 1.92 1.62

Doing 4 electroporations, in order to ensure competent cells made last week work correctly.

Symbol Media Plasmid Cells Why
A LA/Amp PZE-12-CFP My competent MG1655 To get comp cells w/ plasmid
B LA PZE-12-CFP My competent MG1655 To check that my competent cells are alive
C Spec/AMP PZS4-ATTP My competent MG1655 To check that my competent cells transform
D LA/AMP PZE-12-CFP Jeff's competent MG1655 To check that my plasmid works/is correct

Protocol steps on page 1.119 - 1.122 of Molecular Cloning, Volume 1 book

  • used straight glycerol instead of GYT
  • used SOC medium (1 ml per reaction)
  • 1 ul of DNA per experiment (approx 52 ng for my plasmid, did not nanodrop Jeff's spec plasmid)
  • 1.8 kV, 1 second shock

Put in 37 degree shaker w/ the SOC for 1 hour. Spun down, supernatant removed to ~50ul each, resuspended, plated and spun onto correct antibiotic plates w/ LB. Put in 37 degree incubator overnight at 7:45 pm. Will take out tomorrow morning.


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