Bacterial Transformation - Electroporation
Electroporation of freshly plated E. coli, P. aeruginosa, or Salmonella cells
Cell Preparation
1. Streak a single colony on a LB-plate and keep at room temperature.
2. Fill a 1.5 mL eppendorf tube with 500 µL sterile water.
3. Add approximately 3 mg of cells, 3 generous swipes across the plate. It is important
not to scrape the plate since it inhibits transformation.
4. Centrifuge at 14000 rpm 1 minute. Remove supernatant, suspend cells by pipet in 500
µL water.
5. Centrifuge again at 14000 rpm for 1 minute. Remove supernatant, suspend cells in 40
µL of water and keep on ice.
6. Cells are now ready for electroporation.
Electroporation
1. Add 5-50 nG of plasmid DNA and mix gently to ensure homogenous suspension.
2. Transfer DNA/cell suspensions to electroporation cuvettes and keep on ice.
3. After pulsing, immediately add ice-cold LB (or SOC media) to each cuvette.
4. Transfer to culture tubes and incubate for 1 hour at 37°C.
5. Spread on selective plates.
References
Enderle, P.J. & Farwell, M.A., "Electroporation of freshly plated E. coli and P.
aeruginosa cells". Biotechniques. 1998 Dec; 25(6):954-956
Bacterial Transformation - Electroporation
Preparation of Electrocompetent cells
1. Dilute a 5 mL cell culture into 1 L LB and grow to late log phase. Chill cells on ice.
2. Centrifuge cells in GSA rotor @4000 krpm for 15 min. Discard supernatant.
3. Resuspend cells in 500 mL ice cold ddH2O.
4. Centrifuge cells in GSA rotor @4000 krpm for 15 min. Discard supernatant.
5. Resuspend cells in 250 mL ice cold ddH2O.
6. Centrifuge cells in GSA rotor @4000 krpm for 15 min. Discard supernatant.
7. Resuspend cells in 30 mL ice cold ddH2O.
8. Centrifuge cells in SS34 rotor @4000 krpm for 8 min. Discard supernatant.
9. Resuspend cells in 2 mL ice cold ddH2O.
Electroporation
1. Set Pulse Generator to 2.5 kV, 21 µF
2. Add 1 mL DNA in ddH2O to 40 µL Electrocompetent cells.
3. Place cells in electroporation cuvette (2 mm gap) and immediately electroporate.
Decay time should be around 4.6 msec.
4. Immediately add 500 µL ice cold SOC. SOC is 20 mM glucose in SOB:
2.0 %
0.5 %
10.0 mM
2.5 mM
10.0 mM
10.0 mM
Tryptone
Yeast Extract
NaCl
KCl
MgCl2
MgSO4
20.0 g
5.0 g
2 mL
2.5 mL
10.0 mL
10.0 mL
970 mL
BactoTryptone
Yeast Extract
5 M NaCl
1 M KCl
1 M MgCl2
1 M MgSO4
ddH2O
5. Mix gently and incubate on ice for 5 min.
6. Transfer to a culture tube and incubate at 37° for 1 hr with shaking.
7. Plate 50 µL cells and grow overnight.