Heat Shock Transformation with a Water Bath

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 water bath for the heat shock step.

Materials

Reagents

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

Equipment

Water bath set to 42°C
Bunsen burner
Timer
Ice bucket

Procedure

Label and pre-chill centrifuge tube(s) on ice. Ensure the water bath is ready at 42°C. Warm agar plates in the 37°C room.
Under sterile conditions, add 50 µL competent cells per tube and keep on ice.
Pipette 2-5 µL (higher end for larger plasmids) into a centrifuge tube with comp cells. Close tubes and flick gently to mix, but do not vortex.
Incubate tubes on ice for 30 mins.
Using a floating tube rack, heat shock tubes in the 42°C water bath for 30s, then rest tubes on ice for 5 mins.
Under sterile conditions, pipette 950 µL LB or SOC medium (no AB needed) into each tube. This volume can be reduced to concentrate transformants.
Incubate tubes in the 37°C room for 1 hour, preferably on a shaker or rotor. Use one of the designated 50 mL falcon tubes to place microcentrifuge tubes in for a shaker.
(Optional) For experimental tubes and positive controls, perform a 10x dilution in LB or SOC medium in a sterile microcentrifuge tube (100 µL product + 900 µL medium). For negative controls, no dilution is needed.
Under sterile conditions, pipette 100 µL diluted product onto a plate and spread. Keep the agar side down and lid side up.
1. If spreading on the whole plate, use 7-10 glass beads. Carefully deposit the beads in an ethanol-filled beaker (dirty beads beaker).
2. If spreading onto a section of a plate use a glass spreader or loop.
Keep the lid off and allow plates to dry under the flame for 5-10 minutes.
Close and wrap plates, and label appropriately. In addition to section labels, a plate label must contain:
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
Dispose of culture products already inside tubes by closing them tightly and throwing them in the biohazard bin.