E. Transform Competent E.coli cells

The competent E.coli cells are heat shocked to assist the movement of the plasmid DNA into the cell. The heat shock changes the fluidity of the cell membrane allowing the DNA to enter the cell at an efficient rate.

Another method commonly used is electroporation; however, this will not be dealt with here.

Caution: Use sterile technique. You need to take precaution to not contaminate with bacteria or fungi from the air.

E.1 Heat shock *E.coli* cells

  1. Use the cells that you made competent in the previous step. If you prepared them previously and stored them in the -80°C freezer, then remove them from the freezer and thaw them on ice for approximately 30 minutes.
  2. Remove the agar plates from 4°C so that they can come to room temperature. The agar plates should contain the correct percentage of the appropriate antibiotic, see Table 2. The resistance gene for the appropriate antibiotic is carried on the plasmid that will be transformed.
  3. In a sterile microfuge tube, combine 1-5 µL of DNA (which should be between 10-100 pg) into 20-50 µL of competent cells. Mix the contents by flicking the tube with your finger. Incubate on ice for 30 minutes.
  4. Heat shock the transformation tubes by placing the bottom half of the tubes into a 42°C water bath for 45 seconds.
  5. Place the tubes on ice for 2 minutes.
  6. Add 250-1000 µL of LB (without antibiotic) to the bacteria and grow in a 37°C shaking incubator for 45 minutes. This allows the bacteria time to generate the antibiotic resistance proteins so that they can grow once they are plated on the agar plates that contain antibiotic.
  7. Plate some or all of the transformation onto an agar plate that contains the appropriate antibiotic. It is a good idea to plate a small amount (for example, 50 µL) onto one plate and the rest onto another plate. If the transformation was very efficient you are more likely to get single colonies on the plate containing 50 µL of the transformation mixture.
  8. Incubate the plates at 37°C overnight.

Invert the plates, so that if water condensation occurs, the water will collect on the plastic lid, not on the agar.

The next steps on Day 4 and 5 serve to verify that your E.coli bacteria cells were successfully transformed. You will do this by growing a large quantity of potentially transformed bacteria, extracting the plasmid, and verify the presence of the plasmid using agarose gel electrophoresis. This is a quick method, and various other methods are also commonly used; for example, sequencing and enzymatic digestion.