User:JeffreyLau/Notebook/2006-6-15

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2006/06/15

Ligation

  • Since our gel from yesterday showed failure, we ligated DNA extract from David and Peng.
  1. Mixed 6 µL insert (E0241), 2 µL vector (R0010), 2 µL DNA dilution buffer in reaction vial
  2. Added 10 µL ligation buffer
  3. Added 1 µL ligase, mixed
  4. Let incubate 5min at room temperature

Transformation

  1. Mixed 20 µL of ligation product with 30 µL competent cells
  2. Negative control: 2 µL of dH20 with 30 µL competent cells
  3. Positive control: 5 µL of positive control with 30 µL competent cells
  4. Incubate on ice for 20min
  5. Heat shock @42C for 30s
  6. Rest on ice for 2min
  7. Incubate overnight @37C
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