ELECTRO TRANSFORMATION

---

1. thaw electrocompetent cells on ice or RT, keep on ice once thawed

2. because quickness is important in this procedure, gather the following for each sample:

- one 15 ml culture tube with 1 ml SOC; all tubes should be equilibrated and kept in ~37 C H2O in an ice bucket
- one sterile pasteur pipette
- one sterile electroporation cuvette on ice; either 0.1 or 0.2 cm gap cuvettes may be used

3. mix DNA( < 2 ul with low salt content to prevent arcing) with 40 µl cells in an eppendorf tube, keep on ice or alternatively, you may add your DNA to the electro-cuvette (pre-chilled) and then add the electrocompetent cells making sure that the DNA gets mixed well with the cells and skip step 4.

4. transfer the cell/DNA mix into an electroporation cuvette

Note: the gene pulser should already be set properly
- time constant = 4.5 - 5.0 ms
- resistance = 200 W
- capacitance = 25 mFD

for 0.1 cm gap cuvettes, set the volts to 1.7 kV
for 0.2 cm gap cuvettes, set the volts to 2.5 kV

5. pulse the cells once; the voltage display blinks, and the gene pulser beeps

6. quickly transfer 37 C SOC to cuvette, mix by gently pipetting up and down, and transfer SOC/cells back to culture tube; replace in 37 C H2O bath

-At this time you may choose to record the actual volts sent to the cuvette as well as the time constant (these values are determined by pressing the appropriate softkeys on the gene pulser).

7. incubate 30 min. @ 37 C, followed by 30 min. @ 37 C/shaking

8. plate cells* on appropriate antibiotic

* For XL1 Blues, 1.0 ml of the 10 pg pUC 18 control transformation will give ~50 colonies if 5.0 x 10^9 cfu/mg DNA efficiency.

---

Made Feb. 22, 1999; Baker Laboratory

Home | Overview | Highlights | Publications | Members | Information | Links

http://depts.washington.edu/bakerpg