Protocols for the transformation of competent E. Coli cells #

Transformation of chemically competent cells using 96 well PCR plates #

If you need to transform a large number of plasmids I recommend using this protocol as it will allow you to use multi-channel pipetting of your cells and greatly reduce the total protocol time. All the pipetting logistics of this protocol are based on the fact that closely spaced 0.5 ml tubes can be pippetted using the multichannel pippete. It is assumed that competent cells are homemade and stored in 0.5 ml tubes.

Protocol #

  1. Prepare a 96 well PCR plate with an appropriate volume of each of the plasmids you wish to transform. 1 plasmid per well. There should be no more than 5 ul of volume per well.
  2. Thaw competent cells on ice for 10 minutes. Find yourself an empty p1000 tip tack. You will use this rack to hold 0.5 ml tubes will pipetting.
  3. Working in the cold room, place rows of 8 0.5 ml uncapped tubes of competent cells into your empty p1000 tip rack. The tops of the tubes will overlap slightly.
  4. Using a multi-channel pipette, pipette 200 ul of cells into your plasmid containing PCR plate wells. Make sure to be as gentle as possible when pipetting cells. Do not mix up and down.
Since homemade competent cells are dispensed as quickly as possible into 0.5 ml tubes you may find that not all of the cells are at the very bottom of the tube. This will lead to you sucking up significantly less than the full 200 ul. If this is the case just use a regular p200 pipette to top of the wells in your PCR plate that did not get enough cells.
  1. Place cells on ice for 30 minutes. While you are waiting prepare a shallow 42C water bath and 0.5 ml tubes with 350 ul of LB; one tube per transformation. These will be used for recovering your cells after heatshock.
  2. Make sure your PCR plate is well sealed and then place into the water bath for exactly 30 seconds. Remove and allow cells to recover for 5 minutes.
  3. Setup your 0.5 ml tubes in the p1000 tip rack the same way you set up the competent cells. Use the multi-channel pipette to transfer the heatshocked cells from your PCR plate to the recovery tubes.
  4. Shake cells at 37C for 1 hour at 200 rpm.
  5. Plate the recovered cells and incubate overnight at 37C.