Team:Paris Bettencourt/Notebook/Drug Screening/Wednesday 2nd October.html

Target

Wednesday 2^nd October

Race to finish stuff

Grew up 5ml cultures of pSB3K3 and pSB1A3 E0240 from transformations

Plasmid mini preps of pSB3K3 and pSB1A3 E0240

Restriction Digest of CI promoter PCR product with XbaI and PstI

Restriction Digest of Linearized Backbone pSB1C3 with XbaI, PstI, and DpnI

Make new NEB Turbo Chemically Competent Cells: Modified protocol. 4mL of NEBTurbo into 2 x 2mL tubes. Centrifuge at 14000 rpm for 1 minute at 4C. Resuspend in 500uL of 100mM MgCl2 Ice cold. Centrifuge at 14000 rpm for 1 minute at 4C. Resuspend in 500uL of iGEM cc cell buffer Ice cold. Centrifige for 1 minute at 14000 rpm at 4C. resuspend in 100ul of iGEM cc cell buffer Ice cold.

Heat Shock Transformation of pSB1C3 SirA, pSB1C3 FprA, pSB1C3 FdxA into NEBTurbo (new chemically competent cells)

Ligation of CI promoter with pSB1C3 backbone.

Heat Shock Transformation of pSB1C3 CI into NEB Turbo