Heat-shock Transformation of Chemically Competent E. coli
Standard bench procedure for introducing a plasmid DNA construct into chemically competent E. coli by brief heat-shock. The workhorse technique underlying nearly every recombinant DNA workflow, including LibreBiotech's open-reagent production procedures. Written for a novice-to-intermediate audience; assumes basic sterile technique but no prior molecular biology experience.
Version History
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Effective: 2026-04-20Initial release. Atomic technique procedure authored to support LibreBiotech procedure 59 (OpenTaq Cellular Reagent Production) and any future recombinant workflow. Content written from standard molecular biology practice; procedural facts are uncopyrighted field conventions. CC-BY-4.0 for downstream reuse.
Procedure Details
- PC1 (OGTR) / CL1 (HSE) containment required. This procedure produces a genetically modified organism. Must be performed in an OGTR-certified PC1 (AU) or HSE-notified CL1 (UK) facility.
- BSL-1 host. E. coli BL21(DE3) is non-pathogenic, but all live-culture material and waste must be autoclaved before disposal.
- Ampicillin. Wear gloves; avoid skin contact. Autoclave waste containing antibiotic before disposal.
- Heat block at 42°C. Small risk of burns when handling tubes; use tube tongs or a rack; eye protection advised.
- Sterile technique. Every surface touching pre-transformation reagents must be sterile to prevent contamination of your transformants.
What you need ready before starting (allow ~30 min of prep):
- Competent cells thawing on ice in the biosafety cabinet (thaw 10 min; do not refreeze or re-use after thawing).
- Plasmid DNA at a known concentration (ideally 10–100 ng/µL in a sterile 1.5 mL tube; a Nanodrop or Qubit reading confirms this).
- LB or SOC recovery medium pre-warmed at 37°C. SOC is preferred (richer, better recovery) but LB works.
- Selection plates pre-warmed at 37°C for at least 30 min (cold plates reduce colony yield).
- Heat block pre-set to exactly 42°C. Calibrate with an external thermometer if you haven't recently — variation of more than ±1°C meaningfully drops efficiency.
- Ice bucket stocked and staged on the bench.
- Sterile microcentrifuge tubes chilled on ice.
- Sterile spreaders / L-loops and your pipette tip-boxes autoclaved.
Mental model: heat-shock transformation works by a brief membrane permeability pulse that lets DNA enter the cell. Timing and temperature both matter. Too hot or too long kills cells; too cool or too brief and DNA doesn't enter.
- Prep (30 min, hands-on): thaw cells, pre-warm plates and media, calibrate heat block.
- Transformation (90 min, mostly hands-off): DNA addition (1 min) → ice incubation (30 min) → heat shock (45–60 s) → ice recovery (2 min) → outgrowth (45–60 min at 37°C shaking).
- Plating (5 min): spread on antibiotic-selective agar.
- Overnight incubation: 14–16 h at 37°C, plates inverted.
- Next-day result: visible colonies.
-
Microcentrifuge
Centrifugation
Specs: ≥9,000 × g optional
Not required for this technique but commonly staged -
Micropipette
Liquid handling
Specs: P2, P20, P200, P1000
Filter tips; dedicated set if available -
Biosafety cabinet
Safety
Specs: Class II Type A2 or equivalent
All open-culture steps -
Freezer (−20 °C)
Storage
Specs: Standard lab freezer
For plasmid DNA stocks -
Benchtop incubator
Thermal regulation
Specs: 37°C static
Overnight plate incubation -
Heat block
Thermal regulation
Specs: 42°C precise, ±0.5°C
Calibrate with external thermometer before use -
Shaking incubator
Thermal regulation
Specs: 37°C, 225 rpm
Outgrowth phase
-
E. coli BL21(DE3)
(NEB)
C2527IBacterial strain
Qty: 50 µL chemically competent
NEB C2527I or in-house prepared; thaw on ice only once -
pATetO-Taq
(Addgene) Expression plasmid
Qty: 1-10 ng
Or any plasmid of interest; use a known-concentration stock -
Ampicillin
Antibiotic
Qty: 100 µg/mL final on plate
Match antibiotic to plasmid's selection marker (amp, kan, cm, etc.) -
Superior Broth
(Athena Enzyme Systems)
0105Growth media
Qty: 400 µL recovery
LB or SOC equivalent acceptable; pre-warm to 37°C
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
heat_shock_temp_c |
number | — |
42
|
degree Celsius (UO:0000027) | Heat block temperature during the shock step. Standard 42°C; calibrate to ±1°C. Lower temperatures reduce efficiency; higher temperatures kill cells. |
heat_shock_time_s |
number | — |
45
|
second (UO:0000010) | Heat-shock duration in seconds. 45 s for BL21 strains; 60 s for most cloning strains (DH5α, TOP10). Timing precision matters. |
ice_preincubation_min |
number | — |
30
|
minute (UO:0000031) | DNA-cells co-incubation on ice before heat shock. 30 min standard; shorter reduces efficiency. |
recovery_temp_c |
number | — |
37
|
degree Celsius (UO:0000027) | Temperature during outgrowth/recovery step. 37°C standard for E. coli. |
recovery_time_min |
number | — |
50
|
minute (UO:0000031) | Outgrowth duration. 45–60 min typical. Shorter insufficient for resistance marker expression; longer adds no value. |
plasmid_amount_ng |
number | — |
5
|
— | Plasmid DNA amount added to the transformation. 1–10 ng typical; more is wasteful and can yield too many colonies to count. |
competent_cell_volume_ul |
number | — |
50
|
microliter (UO:0000101) | Volume of competent cells per transformation. 50 µL standard; can be halved for cost-sensitive use with proportionally scaled plating volume. |
Procedure Steps (Version 0.1.0)
Remove a 100 µL aliquot of chemically competent E. coli from the −80°C freezer. Place on ice in the biosafety cabinet for 10 minutes to thaw gently. Do not let the tube warm above 4°C and do not refreeze.
While cells thaw, label a sterile 1.5 mL microcentrifuge tube on ice with your strain/plasmid identifier. Pre-cooling the tube prevents the cell membrane from being shocked on contact.
Add 1–10 ng of your plasmid DNA to the pre-cooled tube. The volume of DNA added should be no more than 5% of the total reaction volume (so ≤2.5 µL into 50 µL cells) to avoid lowering the salt concentration needed for DNA uptake.
When cells are thawed, gently transfer 50 µL of competent cells into the DNA-containing tube using a wide-bore tip if available.
Mix by flicking the tube gently 3–4 times. Do NOT vortex — competent cells are fragile and vortex shear kills them.
Incubate the tube on ice for 30 minutes. During this step DNA adsorbs onto the cell surface in preparation for uptake.
Transfer the tube directly from ice to a pre-heated 42°C heat block. Start a timer immediately.
Heat-shock for exactly 45 seconds (for BL21(DE3); use 60 s for DH5α and most cloning strains). Timing is critical — too short reduces uptake, too long kills cells.
Return the tube to ice immediately after heat shock. Incubate on ice for 2 minutes to let membranes reseal.
Add 250–450 µL of pre-warmed (37°C) SOC or LB medium (no antibiotic) to the tube.
Transfer to a 37°C shaking incubator set to 225 rpm. Incubate for 45–60 minutes. This outgrowth step lets the cells begin expressing the antibiotic resistance marker before encountering selection.
Meanwhile, retrieve pre-warmed selection plates (LB + antibiotic agar). Confirm the antibiotic matches the plasmid's resistance marker (e.g. ampicillin for most pUC/pET-derived plasmids, kanamycin for pET-28a, etc.).
After outgrowth, spread 50–200 µL of the culture onto each selection plate using a sterile L-shaped spreader or glass beads. For an unknown transformation efficiency, plate three volumes (50, 100, 200 µL) to ensure at least one plate has countable colonies.
Allow the liquid to absorb into the agar (5–10 min at room temperature in the biosafety cabinet).
Invert the plates (agar side up) and incubate at 37°C for 14–16 hours. Inverting prevents condensation from dripping onto the agar surface.
Remove plates from the incubator the following morning. Count colonies on each plate. Pick 3–5 well-isolated colonies for downstream verification (colony PCR) or immediate liquid-culture growth for glycerol stock preparation.
Seal unused plates with Parafilm and store inverted at 4°C (usable for colony picking for 1–2 weeks).
Record the transformation in LibreBiotech: the Process should reference this procedure_version, link to the Sample records for the competent-cell aliquot used and the plasmid prep, and note the colony count result as a Process annotation.
Expected outcome. 10²–10⁶ colonies on the selection plate for a healthy competent-cell prep + intact plasmid. A "no-DNA" negative control should give zero colonies (confirms the antibiotic selection is working). A known-good positive control plasmid should give a confluent lawn or 10⁵+ colonies (confirms the competent cells are competent).
Picking colonies. Colonies should be well-isolated (ideally 2–4 mm apart), round, and smooth-edged. Irregular "satellite" colonies around larger ones may indicate antibiotic degradation — prefer well-isolated primary colonies.
Storage of plates. Seal inverted plates with Parafilm and store at 4°C. Usable for colony picking for 1–2 weeks; longer storage risks contamination and antibiotic degradation.
Storage of transformants long-term. Pick a verified colony, grow overnight in selective liquid media, and prepare a glycerol stock per the LibreBiotech glycerol-stock procedure. Future batches of this same construct should start from the glycerol stock, not a fresh transformation.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| No colonies on any plate (inc. positive control) | Heat block off-temperature; old competent cells; plate antibiotic too strong | Recalibrate heat block; thaw fresh competent cell aliquot; remake plates with fresh antibiotic |
| Colonies on no-DNA negative control | Antibiotic concentration too low; selection marker not expressing; contaminated stock | Remake plates with correct antibiotic concentration; verify plasmid actually carries the selection marker |
| Confluent lawn (too many colonies) | Too much plasmid added | Dilute plasmid 1:10 and repeat; 1–10 ng is sufficient for most transformations |
| Satellite colonies | Antibiotic-degrading primary colony present | Pick only well-isolated primary colonies; restreak on fresh plate |
References
- Cohen SN, Chang ACY, Hsu L (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. PNAS 69(8):2110–4. DOI paper
- Mandel M, Higa A (1970). Calcium-dependent bacteriophage DNA infection. J Mol Biol 53(1):159–62. DOI paper
- Standard laboratory practice; see standard molecular biology references (e.g. Green & Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed, CSHL Press 2012). book