Electro-competent Bugs

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

Starter:

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

Electroporation:

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