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

Trojan Horse

Anne with Nico, Marguerite and Yonatan

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.

4) Heat shock at 42 °C for 30 seconds.

5) Return to ice for 2 minutes.

6) Add 200 ul LB medium and recover the cells by shaking at 37 °C for 30 min (AmpR)

7) Plate out the cells (10 ul) on Amp LB plates as well as a control (untransformed cells)

8. Incubate at 37 °C. Transformants should appear within 12 hrs.

Result: Colonies (around 10)

Vincent and Aude

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”

Vincent

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