Team:Exeter/Notebook/August18

Notebook (Exeter iGEM 2020)

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Tuesday, 18 August

Lab Work

Anna and Matthew transformed E. coli with the plasmid constructs cloned yesterday. The following protocol was used:

  1. Thaw, on ice, one 100ul aliquot of competent E. coli DH5alpha per transformation, plus one extra as a negative control. Aliquots are labelled 1-12 (refer to yesterday's E. coli well plate plan for which number contains which construct) and 13 (control).
  2. Add 3ul of plasmid DNA, mix by rolling tube.
  3. Incubate on ice for 40 min
  4. Heat shock in 42°C water bath for 2 min, return to ice for 2 min
  5. Add 0.7ml LB medium
  6. Incubate at 37°C, 220 rpm for 60 min
  7. Centrifuge at 8000 rpm for 5 min
  8. Remove 0.5ml of the supernatant
  9. Re-suspend pellet in remaining liquid
  10. Plate out 200 ul onto LB-agar plate with appropriate antibiotic
  11. Incubate at 37°C (no shaking) overnight

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