This is the moment biology becomes engineering: you hand a living cell a new piece of DNA, and it starts following those instructions. The heat-shock method is simple, reliable, and the basis of nearly all molecular biology.
A plasmid is a small loop of DNA that bacteria can carry and read. If that plasmid contains, say, a gene for a green fluorescent protein and a gene for antibiotic resistance, a cell that takes it up will both glow and survive on a plate containing that antibiotic. That antibiotic is your selection: only successfully transformed cells grow.
"Heat shock" briefly makes the cell membrane permeable so DNA can slip inside. We use competent cells — E. coli specially prepared to make this easy.
Thaw competent cells on ice and keep them there. Cold is what keeps the membrane ready to respond to the heat shock — don't let them warm up.
Gently add a few microlitres of plasmid to the cells and swirl (don't vortex). Return to ice for 20–30 minutes so DNA settles against the membrane.
Move the tube to 42 °C for exactly 45 seconds, then immediately back onto ice for 2 minutes. This rapid warm-then-cold is what drives DNA inside.
Add ~250 µL of LB/SOC and incubate at 37 °C for ~1 hour. The cells repair and begin expressing the new resistance gene so they can survive selection.
Spread the culture onto an antibiotic LB plate. Also spread a little onto the plain LB control (proves your cells are alive) and, ideally, streak untransformed cells onto antibiotic (proves selection works — nothing should grow).
Invert and incubate at 37 °C for 16–24 hours. Transformed colonies appear on the antibiotic plate — and if you used a fluorescent plasmid, they'll glow under the right light.
| You see… | Likely cause | Fix |
|---|---|---|
| No colonies at all | Heat shock off, cells warmed, or DNA degraded | Keep cells icy until shock; time 42 °C precisely; use fresh plasmid. |
| Colonies on the no-DNA antibiotic control too | Antibiotic inactive or plates too old | Make fresh selective plates. Store antibiotic cold and use within its shelf life. |
| Growth on plain LB but none on antibiotic | Transformation failed but cells are fine | Recheck DNA amount and heat-shock timing; extend recovery time. |
| A confluent lawn, not colonies | Too many cells plated / contamination | Plate less. Verify sterile technique and that the strain is correct. |
Cold, calcium-treated (competent) cells have membranes primed to take up DNA. The sudden 42 °C pulse creates a thermal imbalance that opens transient pores; DNA that was hugging the membrane slips through. During recovery, the cell transcribes and translates the plasmid's resistance gene, building the protein it needs to survive the antibiotic. Selection then does the sorting for you: only cells carrying your plasmid live to form colonies.
The Genetic Engineering Home Lab includes competent cells, a fluorescent plasmid, selective plates, and everything above — your first real genetic modification.