Heat Shock Transformation with a Thermocycler

Objective

Author(s): Pattarin Blanchard, Ada Jiang

Transform chemically competent E. coli cells to enable uptake of DNA plasmid for replication of genetic material and downstream protein expression. This protocol uses a thermocycler for the heat shock and incubation step.

Materials

Reagents

Sterile PCR tubes
Competent E. coli cells, kept on ice
LB agar plates with appropriate antibiotic (and additives)
Glass beads, glass spreader, or sterile loop
Floating foam tube rack
Sterile microcentrifuge tubes
LB or SOC medium, room-temperature
Plasmids/inserts, thawed on ice
Parafilm strips

Equipment

Thermocycler
Bunsen burner / Flow hood / BSC
Timer
Ice bucket

Procedure

Set up the thermocycler program. These cycles can be combined to one program (set the volume to 52 uL) or they can be set up before the appropriate steps.

Heat shock cycle:

Set volume to 12 µL

4°C for 30 mins
42°C for 10-45 secs, depending on competent cell strain
4°C for 5 mins (Optional) Add an additional 4°C infinite hold step to keep the cells in the thermocycler longer than 5 mins before recovery step.

Recovery cycle:

Set volume to 50-100 μL

37°C for 1 hr
4°C infinite hold

Transformation

Chill PCR tubes on ice and label appropriately. Place agar plates in the 37°C room to warm.
Aseptically add 10 µL competent E. coli to a sterile PCR tube for each transformation desired.
Add 2-5 µL DNA or assembly product for each transformation desired. Close tube and pipette up and down to mix.
1. Consider having one tube with only bacteria (no DNA added) for a negative control.
2. Consider spinning everything down.
Place PCR tubes in the thermocycler and run the heat shock cycle (see above).
Once complete, retrieve tubes and, under a flame, add 88 µL SOC (total volume 100µL) medium to each well. Flick to mix.
1. Spin down the PCR tube before moving on, not enough to pellet bacteria.
Place PCR tubes in the thermocycler and run the recovery cycle (see above).
Under a flame or in a BSC, plate cells on agar plates and leave the lid off for ~5 minutes to dry
1. For one transformation per plate, add 50 µL (Or everything, if possible) of transformed bacteria and spread with 7-10 glass beads. Once finished, carefully “pour” the beads into the dirty beads beaker.
2. If splitting plates into sections (i.e. halves, quadrants, or sixths), add 20 µL of transformed bacteria per section and spread with a spreader or loop.
Once sufficiently dried, close and wrap plates, label appropriately, and place in the 37°C room. The label must have
1. iGEM
2. transformant genotype or shorthand e.g. pRep-HpCA. If a shorthand is used, make sure it is clearly commuicated in the corresponding lab notebook entry
3. Antibiotic (and other additives)
4. Date plated
5. Initials