Electro-competent Bugs

Not all the bacteria are suitable for electrotransformation. Some will lyse during the procedure. (such as: BL21-SI)


0.   Grow o.n. 5 ml LB starter (50ml tube) containing appropriate antibiotics (if required)

1.     Transfer to 400ml LB - with the same antibiotics

2.      Grow until OD600 = 0.5 - 0.7

3.      Transfer to 200ml tube (sterile) on ice

4.      Spin 5'  4000rpm 4˚C

5.      Aspirate the sup

6.      Add 200ml ice cold 10% glycerol

7.      Resuspend gently with pipette

8.      Spin 10'  4000rpm 4˚C

9.      Aspirate the sup

10.  Repeat step 6 to 9 and - aspirate almost all liquid as in step 9.

11.  Resuspend the pellet in the last drops of 10% glycerol

12.  Aliquot 40 ml per tube

13.  Place immediately  in dry ice + EtOH  bath for quick freeze

14.  Store at -80˚C


A.      Thaw aliquot on ice

B.                Add 1mL of DNA (10nM to 100nM) into the thawed tube

C.               Transfer into Electroporation qeuvette (2mm) on ice

D.               Zap in electroporator at: 25mF / 2.5Kvolt  / 200Ohm

E.                Transfer bugs into 0.9ml of warm SOC

F.                1-1 1/2h at shaker 37ºC

G.               plate all after spinning and aspirating the SOC on agar plate w/selection