Team:Paris Bettencourt/Notebook/Tuesday 16th July/Trojan Horse


Transformation of pUC18 (NEB, NEB CC, BL21)

  1. Thaw competent cells on ice.
  2. Place 20 ul of cells in a pre-chilled Eppendorf tube.
  3. add o.5ul of plasmid to the cells
  4. Mix gently by flicking the tube.
  5. Chill on ice for 10 minutes.
  6. Heat shock at 42 °C for 30 seconds.
  7. Return to ice for 2 minutes.
  8. Add 200 ul LB medium and recover the cells by shaking at 37 °C for 30 min (AmpR)
  9. Plate out the cells (10 ul) on Amp LB plates as well as a control (untransformed cells)
  10. Incubate at 37 °C. Transformants should appear within 12 hrs.

Result: Colonies (around 10)

In silico cloning: sRNA gBlocks into pUC18 and litmus28-GFP

Successful with both Gibson assembly and regular cloning.
See file: “in silico cloning trojan horse.geneious”

Streaking Ortiz’s strain ELS-41 and ELS-13 containing Litmus28i_J23115-B0032-GFP and M13K07, respectively.