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Transformation of standard components

  • ligation and transformation of promoter and GFP
  • ligation protocol with the Roche Rapid DNA ligation kit:
  1. 6 μL of insert (E0241), 1 μL of plasmid (R0010 and backbone), and 3 μL of 1x DNA dilution buffer (vial 2) (enough buffer for total volume of 10 μL) into a microcentrifuge tube. (Would ordinarily use 2 μL of plasmid, but the plasmid concentration is about doubled because it combined bands from two gel lanes.)
  2. Mixed ligation buffer (vial 1), and added 10 μL to tube.
  3. Added 1 μL T4 DNA ligase (vial 3).
  4. Incubated on ice for 5 min.
  • transformation:
  1. 30 μL competent cells each into three microcentrifuge tubes.
  2. Ligation mixture, 1 μL water (negative control), and 1 μL pUC 19 (positive control) added to respective tubes.
  3. Incubated the tubes on ice for 20 min.
  4. Heat shocked the tubes at 42°C for 30 s.
  5. Added 200 μL SOC to each tube.
  6. Incubated, with shaking, at 37°C for 1 h.
  7. Plated the mixture on carbomycin plates and incubated at 37°C for 24 h, then stored at 4°C.