Team:Paris Bettencourt/Notebook/Tuesday 16th July/Trojan Horse
From 2013.igem.org
Transformation of pUC18 (NEB, NEB CC, BL21)
- Thaw competent cells on ice.
- Place 20 ul of cells in a pre-chilled Eppendorf tube.
- add o.5ul of plasmid to the cells
- Mix gently by flicking the tube.
- Chill on ice for 10 minutes.
- Heat shock at 42 °C for 30 seconds.
- Return to ice for 2 minutes.
- Add 200 ul LB medium and recover the cells by shaking at 37 °C for 30 min (AmpR)
- Plate out the cells (10 ul) on Amp LB plates as well as a control (untransformed cells)
- 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.