Optimized Bacterial Transformation
Updated 10/5/2024 07:22 PM
Introduction
Materials
- Ice
- Competent bacterial cells
- Plasmids or other vectors
- SOC media
- Eppendorf tubes
- Agar plates
Procedure
Bacterial Transformation
- Prepare tubes and ice bucket: Label 1.5mL tubes with part name or well location. Fill the lab ice bucket with ice, and pre-chill 1.5mL tubes (one tube for each transformation) in a floating foam tube rack.
- Thaw competent cells on ice: This may take 10-15 min for a 200µL stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
- Pipette 50µL of competent cells into 1.5mL tube: 50µL in a 1.5mL tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice.
- Pipette 2µL of assembly reaction into 1.5mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
- Close 1.5mL tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
- Heat shock tubes at 42°C for 45 sec: 1.5mL tubes should be in a floating foam tube rack. Place the floating rack in a water bath set to 42°C to ensure the bottoms of the tubes are submerged.
- Incubate on ice for 5 min: Return transformation tubes to the ice bucket and let them recover for 5 minutes.
- Pipette 950µL SOC media to each transformation: SOC should be warmed to room temperature before use. Check for contamination.
- Incubate at 37°C for 1 hour, shaking at 200-300 rpm.
- Pipette 100µL of each transformation onto LB agar plate with the appropriate antibiotic: Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
- Plate remaining culture: Spin down cells at 6800g for 3 mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto LB agar plate with the appropriate antibiotic. Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
- Incubate transformations overnight (14-18 hr) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
Transformation is the process of introducing foreign DNA (often a recombinant plasmid ligated with the gene of interest) into bacterial cells. In order to take up foreign DNA, the bacterial cells must first be made competent. This can be done by treating bacteria with CaCl2 prior to transformation, as CaCl2 destabilizes the bacterial cell membrane, forming small pores through which the foreign DNA can be taken up. It also allows DNA to bind to the bacterial cell wall. There are two methods of transformation: heat shock, in which the competent bacterial cells and plasmids are subjected to brief heat shock, opening up the pores in the bacterial cell membrane, and allowing it to take up the plasmid, and electroporation, in which the competent bacterial cells and plasmids are subjected to electrical shock instead of heat shock. In order to select for the bacteria that have successfully been transformed and taken up the plasmid, the bacteria are grown on agar plates with growth medium to which an antibiotic is added. Plasmids can carry one or more resistance genes for antibiotics, such as ampicillin, kanamycin, and chloramphenicol. Successfully transformed bacteria will express the antibiotic resistance gene, allowing the bacteria to resist the antibiotic when exposed to it, while bacteria which have not been successfully transformed will not express the antibiotic resistance gene and hence will not survive.