Team:Toulouse/Experiments

iGEM Toulouse 2015

YETM 500mL	TFB1 200mL	TFB2 200mL
2.5g Yeast Extract 10g Tryptone 5g MgSO4.7H2O Adjust pH to 7.5 with KOH For Plates: add 7.5g of Agar	0.59g KOAc 2.42g RbCl 0.29g CaCl2.2H2O 1.98g MnCl2.4H2O Adjust to pH 5.8 with 0.2 M acetic acid Add dH2O to 200mL Filter sterilize Store refrigerated at 4°C	0.42g MOPS 2.21g CaCl2.2H20 0.24g RbCl 30g Glycerol Adjust to pH 6.5 with KOH Add dH2O to 200mL Filter sterilize Store refrigerated at 4°C

1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C
2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.
Do not vortex cells at any time after this point in the procedure
3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C

4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant
Do not allow cells to warm above 4°C at any time in this procedure
6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
7. Incubate cells on ice for 5min
8. Repeat step 5
9. Resuspend cells in 2mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
10. Incubate cells on ice for 15 minutes
Cells may be used for transformation or frozen

To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice)
Freeze immediately in liquid nitrogen
Store cells frozen at -80°C

References