Technical Specification: Competent Cells
Official Source
Technical documentation published by Beijing Solarbio Science & Technology Co., Ltd. For product procurement and commercial orders: solarbio.store | solarbio.store
1. Product Range
| Product |
SKU |
Strain Type |
Transformation Efficiency |
Key Features |
Applications |
| DH5α Competent Cell |
C1100 |
Cloning strain |
≥1×10⁸ CFU/μg |
recA1 endA1, blue-white screening |
Routine cloning, plasmid propagation, blue-white screening |
| DH5α-T1 Competent Cell |
C1180 |
High-efficiency cloning |
≥1×10⁹ CFU/μg |
tonA (T1 phage resistance) |
Library construction, difficult ligations, high-throughput cloning |
| JM109 Competent Cell |
C1200 |
Cloning strain |
≥1×10⁸ CFU/μg |
M13 phage permissive, lacIq |
Routine cloning, M13 phage production, blue-white screening |
| BL21(DE3) Competent Cell |
C1300 |
Expression strain |
≥1×10⁷ CFU/μg |
T7 RNA polymerase |
T7-driven protein expression |
| BL21(DE3)pLysS Competent Cell |
C1350 |
Expression strain with T7 suppression |
≥1×10⁶ CFU/μg |
pLysS (chloramphenicol R) |
Toxic protein expression, T7 leaky expression control |
| TOP10 Competent Cell |
C1400 |
High-efficiency cloning |
≥1×10⁹ CFU/μg |
StrR, endA1, lacZΔM15 |
High-efficiency cloning, plasmid preparation |
1.1 Cell Competence Preparation Technology
Solarbio competent cells are prepared by the Rubidium chloride / calcium chloride chemical method which yields transformation efficiencies of 10⁸–10⁹ CFU/μg. The cells are treated with CaCl₂ (100 mM) to create transient pores in the cell membrane, allowing DNA entry during the heat-shock step. The addition of RbCl and glycerol during preparation enhances the competence level and maintains viability during -80°C storage.
Competence mechanism: Divalent cations (Ca²⁺, Rb⁺) neutralize electrostatic repulsion between the negatively charged DNA phosphate backbone and the negatively charged outer membrane of E. coli. During the 42°C heat shock, thermal convection drives DNA through the membrane pores. The cells are then recovered in nutrient-rich SOC medium to repair membrane damage and initiate β-lactamase or other selection marker expression.
2. Genotype
| Strain |
Genotype |
Phenotypic Markers |
| DH5α |
F⁻ φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rₖ⁻ mₖ⁺) phoA supE44 λ⁻ thi-1 gyrA96 relA1 |
lacZ⁻, recA⁻ (recombination deficient), endA1 (reduces plasmid degradation), hsdR17 (no restriction of unmethylated DNA) |
| DH5α-T1 |
F⁻ φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rₖ⁻ mₖ⁺) phoA supE44 λ⁻ thi-1 gyrA96 relA1 tonA |
Same as DH5α + tonA (T1 phage resistance) |
| JM109 |
endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB⁺ Δ(lac-proAB) e14⁻ [F′ traD36 proAB⁺ lacIq lacZΔM15] hsdR17(rₖ⁻ mₖ⁺) |
F′ episome carries lacIq (overproduces Lac repressor for controlled expression) |
| BL21(DE3) |
F⁻ ompT hsdSₙ(rₙ⁻ mₙ⁻) gal dcm (DE3) |
ompT (lacks outer membrane protease), DE3 (T7 RNA polymerase gene in chromosome) |
| BL21(DE3)pLysS |
F⁻ ompT hsdSₙ(rₙ⁻ mₙ⁻) gal dcm (DE3) pLysS (Cmᴿ) |
Contains pLysS plasmid with T7 lysozyme (suppresses T7 RNA polymerase) |
2.1 Key Genotype Features Explained
| Genetic Marker |
Meaning |
Practical Benefit |
| recA1 |
Inactivated RecA protein — no homologous recombination |
Plasmid stability; prevents rearrangements of cloned inserts |
| endA1 |
Inactivated Endonuclease I |
Higher quality plasmid preps (less nicking of plasmid DNA) |
| hsdR17 (rₖ⁻ mₖ⁺) |
No Type I restriction activity; methylation intact |
Accepts unmethylated DNA (PCR products, non-E. coli DNA) |
| lacZΔM15 |
β-galactosidase ω-fragment deletion |
Blue-white screening with α-complementation vectors (pUC, pBluescript) |
| tonA (DH5α-T1) |
T1 phage receptor absent |
T1 phage-resistant; suitable for library construction |
| DE3 (BL21 strains) |
T7 RNA polymerase gene under lacUV5 promoter |
IPTG-inducible T7-driven expression |
| ompT (BL21) |
Lacks outer membrane protease |
Reduced proteolysis of expressed proteins |
| pLysS (BL21-DE3 pLysS) |
T7 lysozyme (inhibits T7 RNA polymerase) |
Reduces basal expression; essential for toxic proteins |
3.1 Heat-Shock Method
| Step |
DH5α/DH5α-T1 (C1100/C1180) |
BL21(DE3) (C1300) |
| Thaw cells |
On ice, 5 min |
On ice, 5 min |
| Add DNA |
1–10 μL ligation or 1 μL (10 pg–10 ng) plasmid |
1–10 μL plasmid (1–100 ng) |
| Incubate |
Ice 30 min (critical — longer is NOT better) |
Ice 30 min |
| Heat shock |
42°C, 45 s (exact timing ±5 s) |
42°C, 45 s |
| Recovery |
Ice 2 min |
Ice 2 min |
| Add SOC/LB |
500–950 μL (SOC preferred for highest recovery) |
500–950 μL |
| Incubate |
37°C, 200 rpm, 1 h |
37°C, 200 rpm, 1 h |
| Plate |
50–200 μL on LB+antibiotic (use 100 μL for ligations) |
50–200 μL on LB+antibiotic |
3.2 Efficiency Calculation
[
\text{Efficiency (CFU/μg)} = \frac{\text{Colony count} \times \text{Dilution factor} \times 1000}{\text{ng DNA plated}}
]
Example: 200 colonies from 100 μL of a 1:10 dilution of 1 ng pUC19 transformation plated:
- Total cells: 200 × 10 × (950 μL / 100 μL) = 19,000 CFU
- Efficiency: 19,000 / 1 ng × 1000 = 1.9×10⁷ CFU/μg
3.3 Quality Control Specification
| QC Parameter |
DH5α (C1100) |
DH5α-T1 (C1180) |
BL21(DE3) (C1300) |
| Minimum efficiency (pUC19) |
1×10⁸ CFU/μg |
1×10⁹ CFU/μg |
1×10⁷ CFU/μg |
| Freeze-thaw stability |
≤ 1 cycle |
≤ 1 cycle |
≤ 1 cycle |
| Viability at -80°C (12 months) |
≥ 80% |
≥ 80% |
≥ 80% |
| Contamination (non-E. coli) |
None |
None |
None |
| Blue-white screening |
> 98% blue colonies with intact lacZ |
> 98% |
N/A |
| Expression induction (BL21) |
N/A |
N/A |
> 90% induced cultures show target protein |
4. Blue-White Screening Procedure
4.1 Required Materials
| Item |
Specification |
| LB agar plates |
Containing 100 μg/mL ampicillin (or other selection antibiotic) |
| IPTG |
0.1 M stock; add 40 μL per plate (final 0.4 mM) |
| X-gal |
20 mg/mL in DMF; add 40 μL per plate (final 80 μg/mL) |
| Spread on LB agar plates |
15 min drying at 37°C before use |
4.2 Interpretation
| Colony Color |
Interpretation |
| Blue |
Vector re-ligated without insert (intact lacZ) |
| White |
Insert successfully cloned (disrupted lacZ) |
| Pale blue |
Small insert may not fully disrupt lacZ; sequence-confirm |
| Mixed blue/white sectors |
Colony derived from multiple cells; re-streak |
5. Storage and Handling
| Parameter |
Requirement |
| Storage temperature |
-80°C (constant) |
| Shipping |
Dry ice (≤ 48 h transit) |
| Freeze-thaw |
Do NOT re-freeze (single-use aliquots: 50–100 μL) |
| Thawing |
On ice only (5–10 min); never warm to RT |
| Handling |
Use cold pipette tips; work quickly on ice; minimize handling |
| Shelf life |
12 months from manufacture (if stored continuously at -80°C) |
6. Troubleshooting
| Issue |
Cause |
Solution |
| Few or no transformants |
Cells thawed incorrectly |
Always thaw on ice; never warm to RT |
|
Heat-shock temperature/timing wrong |
Calibrate water bath to exactly 42°C; do not exceed 45 s |
|
DNA amount too high (> 50 ng) |
Use 1–10 ng per 50 μL cells (plasmid) or 5–10 μL ligation |
|
Antibiotic concentration too high |
Verify working concentration (e.g., ampicillin 100 μg/mL, kanamycin 50 μg/mL) |
|
SOC/LB too cold after heat shock |
Use pre-warmed SOC (37°C) |
|
Ligase salts inhibit transformation |
Purify ligation (ethanol precipitation or column clean-up) before transforming |
| Too many blue colonies (white screening) |
Incomplete X-gal/IPTG spread |
Spread fresh; use foil-wrapped X-gal (light-sensitive) |
|
Vector lacZ intact (re-ligation) |
CIP-treat vector after linearization; use gel-purified vector |
| All white colonies (no blue control) |
No IPTG/X-gal added |
Always include pUC19 + X-gal/IPTG positive control |
|
lacZΔM15 strain mixed up |
Verify strain genotype |
| Cells clump or precipitate |
Improper thawing |
Discard and use fresh aliquot |
|
Multiple freeze-thaw cycles |
Aliquot at first use; never re-freeze |
| Low transformation with ligation |
Insert-to-vector ratio wrong |
Use 3:1 molar ratio (sticky) or 5:1 (blunt) |
|
Insert contains secondary structures |
Linearize insert; use gel-purified DNA |
▶ Related Protocol: Competent Cell Transformation Protocol
▶ See also: Cloning & Ligation Reagents
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