3 June 2009

From 2009.igem.org

Aim

  • Transformation

Materials:
•TE Buffer
•Plasmids from Kit Plates 1 & 2
•Competent E.coli
•NZY Medium
•Ampicllin, Kanamycin, Tetracyclin
•Agar Plates
•Eppendorf (labelled)

Methods：

  • Transformation Protocol
  1. Hydrate plasmids by 10µL TE buffer.
  2. Add 5µL plasmid solution into labelled eppendorf, together with 15µL TE buffer. (Total Vol = 20µL)
  3. Take 5µL from the 4-fold dilution in step 2, add into the competent E.coli.
  4. Incubate on ice for 30 min. (minimum) [Actual incubation time in lab: >1 hr]
  5. Incubate at 42°C for 45 sec.
  6. Incubate on ice for 2 min. (minimum)
  7. Add 300µL of NZY medium.
  8. Incubate with shaking at 37°C for 1 hr.[Actual incubation time in lab: 40 min]
  9. Spin the incubated E.coli suspension for 3min at maximum speed.
  10. Discard about half of the liquid, and then re-suspend the E.coli solution.
  11. Spread the suspension onto selective agar plates.
  12. Incubate at 37°C overnight.
  • Making Agar Plates
  1. Defreeze agar in microwave oven, 1 min interval.
  2. Check and shake until completely melting.
  3. Add 100µL antibiotic into 50ml agar when it cools down to 50°C.
  4. Pour the plates.
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