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From 2013.igem.org

Glycerols of the overnight cultures of the transformation.

• sT002 : MG1655 pCOLADuet-1
• sT003 : MG1655 pACYCDuet-1

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

Making electroconpetent cells stock

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