29 June 2009

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Aim:

  • Transformation of new (super)parts
  • Incubation

Materials:
•TE Buffer
•Plasmids from Kit Plates 1: R0040; Q04400
•Competent E.coli
•NZY Medium
•Ampicllin, Kanamycin, Tetracyclin
•Agar Plates
•Eppendorf (labelled)

Methods:

  • Tranformation Protocol 2:
  1. Using the puch tool, puch out the appropriate DNA. Clean the tool between punches.
  2. Soak the spots in 5µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.
  3. Add 2µL of DNA in TE to 50µL of competent cells (TOP10)
  4. Allow the DNA and competent cells to sit on ice for 30 minutes
  5. Heat shock at 42ºC for 60 sec in water bath.
  6. Recover on ice for 5 min.
  7. Add 300 µL NZY medium.
  8. Incubate at 37ºC for 2 hr while the tubes are rotating.
  9. Centrifuge and leave about 250 µL liquid; hence, resuspend the E.coli suspension.
  10. Plate 100µL on an LB plate with the appropriate antibiotic.
  • Incubation:
  1. Pick new colonies from plate 15 & 16.
  2. Incubat with shaking overnight.
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