(Modification from Lisa Vaillancourt Lab)
1. We use TOP10F’ cells (Electrocomp Kits, INVITROGEN cat C6665-55, Is in the -80C. READ CAREFULL Kit information, print out in the lab folder “TECHNICAL INFORM”
Remove a tube of frozen competent cells (e. coli) from the –80C. Thaw the cells slowly on ice. Mix gently to resuspend (do not pipet up and down), and return to the ice. Only thaw the cell you need. do not leave them on ice for an extended period of time. DO NOT re-froze them.
2. Pre-chill required number of electroporation vials on ice. Label tops. (is very important that the cells and cuvettes remain cold until after the pulse is applied).
3. Mix 1ml DNA and 20 ml of chilled cells in cold microfuge tubes. Transfer to chilled electroporation vials, knock sharply on the desk to remove any bubbles. Return vials to ice.
4. Set up the gene pulser apparatus (room 305. 3 floor. Turn on the apparatus and check the settings:
• Voltage, 2.5kV
• Capacitor, 25uF
• Pulse controller setting: 200 ohms
Set the indicator to read the time constant. If all goes well, this should be around 4.5-4.7 for each sample.
5. Place the electroporation vial containing the DNA and bacterial cells into the chamber (dry off the bottom with a kimwipe, first), slide it into place so that contact is made with the electrodes, then push both buttons on the apparatus at the same time, until the buzzer sounds. If sparks then change the voltage for next sample to 1.7 kV.
6. AS QUICKLY AS POSSIBLE: slide the electroporation cell out of the chamber, take off the lid, and add 480 ml of chilled SOC medium to the cuvette. Mix briefly, then return the cells to the ice until you have finished electroporating all your samples.
7. Transfer samples to 15ml tubes, incubate the tubes for 1 hr at 37 C on the shaker at 225rpm (loose cups)
8. Spread 100 ml. of the cells onto LBamp (LB + ampicillum) plates (prewarmed), incubate upside-down at 37 C overnight. Save the rest in the 4C fridge.
Optional, if the efficiency of transformation is very low (like plasmid rescue). Then after step 7continue with 7.1
7. 1. Transfer samples to 1.5 ml microfuge tube. Spin 5 min at a very low setting (like 4000 rpm). Tip the supernatant. Resuspend cell in LB broth into 200 ml. continue with step 8.
For appropriate control check the Electrocomp Kit information from INVITROGEN.