There are two common protocols for transforming E. coli with in vitro DNA: heat shock and electroporation. Note that it’s always a good idea to confirm your transformants both phenotypically and molecularly (with PCR).

Heat Shock Transformation

  1. Grow an overnight culture of the E. coli strain you want to transform in LB medium at the temperature (usually 37 C) you will use to grow the transformed strain.
  2. The morning of the transformation, transfer 100 uL of the overnight culture into 9.9 mL of fresh LB. Place back in incubator with 120 rpm shaking.  Grow for approximately 3 hours.  Less time is okay as long as the culture is noticeably more turbid than it was when you inoculated; more not so much.
  3. Meanwhile, turn on a hot block and set it to 42 C.  Fill the tube slots with water to create a “mini water bath”.
  4. Get a bucket of ice and place a tube of sterile 0.1M CaCl2 and one sterile 1.5 mL Eppendorf tube for each transformation on ice to chill. Make sure Eppendorf tubes are pre-labeled as it is hard to write on cold tubes with a sharpie.
  5. Place 3 agar plates of the medium you will use to select for transformants in an incubator at the selection temperature.
  6. Set the centrifuge to pre-chill to 4 C.
  7. When the culture is ready, centrifuge the whole thing at 5000 rcf for 5 minutes at 4 C.
  8. Pour off the LB media and remove the last few drops with a 100 uL pipet tip.
  9. Resuspend the pellet in ice-cold 0.1M CaCl2. Use 50 uL for each transformation you want to do. Transfer 50 uL of resuspended cells to each chilled Eppendorf tube and return to ice.
  10. Place 2 uL of the appropriate DNA solution into each tube.  Chill DNA-cell mixture for 30 minutes.
  11. Place tubes in the 42 C hot block for EXACTLY 60 seconds and IMMEDIATELY return to ice afterwards.
  12. Move tubes out of ice to a normal tube rack after a couple of minutes. Add 450 uL room-temperature LB to tubes. Use SOC media if you’re working with an expression vector (e.g. pBAD vectors).
  13. Shake tubes horizontally at 250 rpm for 1 hour at the selection temperature.  Use 1.5 hours if selection at 30 C or 2 hours if selecting at room temperature.
  14. Plate on selective medium.  I usually put 5 uL, 25 uL, and 200 uL on three different plates.  Incubate plates at selective temperature overnight and put the rest of the transformation mixture in the fridge.