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