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
- 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.
- 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.
- Meanwhile, turn on a hot block and set it to 42 C. Fill the tube slots with water to create a “mini water bath”.
- 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.
- Place 3 agar plates of the medium you will use to select for transformants in an incubator at the selection temperature.
- Set the centrifuge to pre-chill to 4 C.
- When the culture is ready, centrifuge the whole thing at 5000 rcf for 5 minutes at 4 C.
- Pour off the LB media and remove the last few drops with a 100 uL pipet tip.
- 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.
- Place 2 uL of the appropriate DNA solution into each tube. Chill DNA-cell mixture for 30 minutes.
- Place tubes in the 42 C hot block for EXACTLY 60 seconds and IMMEDIATELY return to ice afterwards.
- 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).
- 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.
- 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.