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


Trojan Horse

4th July


  • 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