Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 4th July.html

Trojan Horse

4^th July

Aude

• Glycerols of the overnight cultures of the transformation

• sT002 : MG1655 pCOLADuet-1

• sT003 : MG1655 pACYCDuet-1

Aude and Clovis

Making stocks of electro competent NEB turbo

Prepare 10% glycerol solution

Put on Ice 1L of Sterile water, 10% glycerol solution and 10 falcon tubes

Cool down centrifuge to 4°C

Prepare a rack with 50 microtubes at -80°C

• Dilute in 500 mL LB (in a 1000mL flask) 500 ul of NEB turbo pre cultured

• Check the OD until it reached 0,5

• Put 500mL cultures in 10 50mL falcon tubes in ice

• Wait for 20 minutes for the culture to cool down

• Centrifuge at 3400 rpm 4°C for 10 minutes

• Take out liquid and resuspend in 50 mL ice cold water (resuspend content of two tubes in one tube)

• Centrifuge 10 minutes, 4°C , 3400 rpm

• Take out liquid and resuspend in 25 mL ice cold water

• Centrifuge 10 minutes, 4°C, 3400 rpm

• Resuspend all the content of the tubes in 20mL ice cold glycerol

• Centrifuge 10 minutes, 4°C, 3400rpm

• Resuspend in 5mL ice cold 10°C glycerol solution

• Aliquot in 50 tubes (100uL per microtubes)

• Store at -80°C