Transforming electrocompetent cells

Posted onAuthorBaradwaj G Ravi

Transforming electrocompetent cells

 

  • Thaw aliquot(s) of electrocomp cells (green tubes, in tip box in the front of -80 "Competent Cells" box) on ice (5-10 minutes or until thawed). Spin down in nanofuge.

  • Chill cuvettes on ice (5-10 minutes or until cold). The cuvettes must be ice cold for maximum efficiency.

  • Add DNA (1-2 uL of Gibson mix or 0.5 uL supercoiled vector) to cells without mixing or introducing bubbles (1st stop on pipet only).

  • Transfer cells to cuvette gap without introducing bubbles (e.g. with a P20, twice); it's fine to leave 1-2 uL of cells behind or outside the gap.

  • Tap sides of cuvette to distribute cells horizontally in gap and inspect for bubbles.

    • If there are bubbles, remove them by dragging them up with a pipet tip, by pipetting them out, or by tapping down the cuvette vigorously

  • Take up 1 mL LB in P1000 and leave hanging next to a flame. Get out a 1.7 mL sterile tube.

  • Turn on electroporator and press blue "Measurements" button twice until "ms" is displayed.

  • Dry sides of cuvette thoroughly with a Kimwipe. Remove cap from cuvette. Tap down ~3x. Place in electroporator with nub facing inward. Slide in completely. Shock and note the pulse time value.

    • Cloning: 2.8 ms or less -> probably no colonies; otherwise LOWER values give more colonies.

    • Vector: Anything less than 5.8 ms will work fine.

    • Visible spark accompanied by popping sound: all cells dead; clean out cuvette and start over with new cuvette (and possibly less DNA)

  • Immediately resuspend cells in LB and transfer to 1.7 mL tube. Recover at 37 degrees with shaking for 30-60 minutes (can truncate or omit this step if selecting with Cb).

  • WASH CUVETTES​​ with water then a couple of squirts of EtOH. Put in "Used cuvettes, washed".

  • Get out plates and let warm to room temp (optional).

  • Spin cells down for 30 sec at max speed; decant most of supernatant; resuspend and plate.

    • Streak out (spread evenly across 1/2 of plate then dilute serially by streaking 3-4x across the rest) if you expect tons of colonies (supercoiled vector); bead if you expect some or many (cloning).

  • DON'T FORGET TO WASH THE CUVETTES.

 

Ben

5/24/12