Day1
-
100
mM MgCl2
- 100 mM CaCl2 with 15 % glycerol)
*1 peak a single colony from LB-agar or scrape the frozen surface of a glycerol stock
Day2
- Inoculate 5ml of the pre-culture in 500ml of LB in a 2liter shaking flask (dilution 1:100) and growth at 180rpm- 37°C up to OD(600nm) about 0,4-0,6;
-
Centrifuge
in a sterile tube for 10 minutes at 2500 g - 4 °C
- Re-suspend the cells with 50 ml of 100 mM MgCl2 COLD
*2 From now all the steps have to be performed in ICE and centrifuge need to be COLD, because an heating of the sample may result in decrease of competence level.
-
Incubate
in ICE for at least 30 minutes (longer incubation are not an issue)
-
Centrifuge
at
-
Discard
the surnatant and transfer the tube containing the cells in ICE.
-
Re-suspend
the cells with 10 ml of
n.b: Vortex can be used for facilitate the cell resuspension with the recommendation to take short steps and not to keep cells out of the ice for too long.
-
Spilt
the cells in aliquots in 1.5ml Eppendorf COLD (100-200ul/Eppendorf)
To maximize the transformation efficiency: If you have DRY ICE available, Cool down empty Eppendorf tubes in dry ice is preferable to froze immediately the cells before storing at -80°C. Alternatively, cold down the Eppendorf in ICE and just transfer the spitted cells on the -80°C.
The cells competency can be verified performing a transformation test with 20pg of puc19
Generally, BL21 à show an efficiency of about 10^6 cfu/ml à about 20 colonies
DH5alpha. DH10B, Top10, MACH1 à shows higher efficiency, about 5-10X10^7 cfu/ml à 50-100 colonies
In case you would like to produce competent cells free of animal components you can use YGR or Enpresso B animal free (http://enpresso.de/en/products/as) culture media.
More information about YGR and Enpresso are available at the following link:
ProteoCool n°7: High cell density E.coli protein expression using Enpresso B medium
ProteoCool n°32 High cell density E.coli protein
expression using YGR medium
