E coli Chemical Transformations

Transformation of competent E. coli cells

We use OneShot TOP10 Chemically competent E. coli cells (comparable to DH5α) or home-made competent cells that are to be found in the -80°C freezer (Parsch freezer).
When using commercial cells, for each transformation, we use 25μl. Each tube in the -80 can thus be used for 2 transformations.

Do transformation using the long version of the manufacturer’s protocol:

• Thaw the cells on ice (and split over 2 tubes to get 25μl cells, do not pipette more than once as cells are very sensitive).
• Add DNA to the cells:
  • 5 μl PCR product of an sgRNA cloned linear plasmid; or
  • 5 μl ligated DNA; or
  • 2-5 μl Gibson assembled plasmid; or
  • 2-10 μl Golden Gate assembled plasmid.
• Flick and place back on ice for 30 min
• Heatshock the cells for 60sec at 42°C
• Place back on ice for 2 min
• Add 250μl 37°C SOC or LB (without antibiotic)
• Incubate these tubes with shaking at 37°C for 1 hour for ampicillin marker or 75-90min for kanamycin.
• Preheat LB plates with appropriate antibiotic (50 μl Ampicillin 100mg/ml stock or 25 μl Kanamycin 50mg/ml stock) to 37°C.
  • add 40 μl X-Gal (freezer 7 top drawer) when blue white screening (e.g. using TOPO cloning) is required
• Spread the 100 μl cells on the preheated LB+amp plates and incubate overnight at 37°C.

  the rest of the cells can be left in the fridge to plate out the next day if needed (e.g. when too many or too few colonies were obtained).

• After one day the plates should show colonies