ProteoCool Pills#3: Homemade E.coli chemically competent cells

                               

                                         

                               For preparation of 10ml competent cells from 500ml of culture

Day1

 Preculture: Inoculate the *cells in 10 ml of LB (in a 50ml falcon) O/N at 180rpm- 37 °C

 Store in fridge at 4°C the solutions (filtered at 0.22uM) :

-        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

 -        Discard the surnatant (LB) and transfer the tube with the cells in ICE*².

-        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 2500 g for 10 min a 4°C

-        Discard the surnatant and transfer the tube containing the cells in ICE.

-        Re-suspend the cells with 10 ml of 100 mM CaCl2 with 15 % glycerol (COLD)  

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