Bacterial Transformation

Rationale

Transformation stands for the insertion of a plasmid molecule in a bacterial cell, usually by means of a temperature or electrical shock. The shock is supposed to destabilize the membrane of the bacteria, allowing an influx of the negatively charged DNA molecules.

After the shock there is a recovery step where we place the bacteria in non-selective media for it to produce some antibiotic resistance protein before going in selective media. If we placed it in selective media right away, some antibiotics would stop growth even if the cells have our desired DNA.

To execute this protocol you first need competent cells: a preparation that makes the cells prone to receive DNA. Some bacterial species present natural transformation, where they don't need this extra preparation.

Heat-shock method

Before starting this protocol, it's useful to set up either a bucket of ice and a heat-block at 42°C, or a thermocycler with the appropriate program. Pre-preparing LB-Agar plates with antibiotics is also good although this can be done in the recovery step.

Take an aliquot of competent cells from a -80°C freezer and thaw on ice (~10 min).
Separate your competent cells in aliquots of the desired volume (often 50 uL)
Add DNA (variable volume) and incubate in ice for 20 min
Quickly move to 42°C for 45s. Timing is important, so try not to deviate from this.
Move back to ice while you prepare the next step
Recovery: Place cells in 1mL of LB or SOB at 37°C for 1h (can be reduced to up to 20 min)
Centrifuge the cells (E.coli: ~10K G for 5min) and discard ~900uL of media, then ressuspend
Plate cells in selective-media plates and leave at 37°C for 12-16h

Alternatively, the ice incubation and heat-shock can be done in a thermocycler with PCR tubes instead of manually (that's especially relevant for big rounds of 20+ transformations). Pay attention to the ramp speed of your thermocycler to determine the timings, but overall follow the following program:

22 min: 4°C
45 s: 42°C
- : 4°C

The two extra incubation minutes are to allow for you to start the program without the samples, then transfer them into the machine once it's at 4°C.

Electroporation method

Plating methods

Plating cells is normally done with L-spreaders or glass beads to move the cell-containing liquid around the plate, but it can also be done by just dropping a larger amount of liquid and swirling the plate to spread it, then allowing some time with the plate open for it to dry.