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| Product Name | Calcium Phosphate Transfection Kit, Classic HEK293 Method |
| Catalog No. | ATR-CTR-0027 |
| Description | Calcium phosphate co-precipitation transfection kit based on the classic Graham and van der Eb method, optimized for high-efficiency transient and stable transfection of HEK293, HEK293T, and other adherent cell lines. The method involves slowly forming a fine calcium phosphate-DNA co-precipitate by mixing calcium chloride with DNA in phosphate-buffered saline. The precipitate particles adhere to the cell surface and are internalized by endocytosis. DNA is released from endosomes and transported to the nucleus for expression. The kit provides optimized 2× HBS buffer (HEPES-buffered saline) with precisely controlled pH (7.05-7.12) that is critical for consistent, fine precipitate formation required for high transfection efficiency. |
| Intended Use | Transient and stable transfection of HEK293, HEK293T, HEK293FT, and other adherent cell lines for recombinant protein production, lentivirus/retrovirus packaging, and reporter gene assays. |
| Principle / Technology | Calcium phosphate and DNA co-precipitate in phosphate buffer at precisely controlled pH (7.05-7.12); fine precipitate particles adsorb to cell surface via calcium-dependent interactions; endocytotic uptake of particles; DNA escapes endosomes and enters nucleus; slow, dropwise mixing of DNA-CaCl2 into 2× HBS is critical for optimal precipitate formation. |
| Detection Method | Mix DNA (10-50 µg) with CaCl2 and water; add mixture dropwise to 2× HBS while vortexing or bubbling air; incubate 15-20 min at RT for precipitate formation; add precipitate dropwise to cells; incubate 4-16 hours; replace medium; assay after 24-72 hours. |
| Sample Type | Adherent HEK293, HEK293T, HEK293FT, HEK293A, and selected other epithelial and fibroblast cell lines. |
| Performance Range / Specifications | Transfection efficiency: 50-90% for HEK293T cells with EGFP reporter plasmid; stable transfection frequency: 10^-3 to 10^-4 when using linearized plasmid with selection marker. |
| Sensitivity / LOD | Transgene expression detectable at 24-48 hours; stable colonies visible within 10-14 days of antibiotic selection. |
| Specificity | Works best with HEK293 lineage cells; efficiency is cell line dependent; less efficient for suspension, hematopoietic, or primary cells; method is sensitive to precipitate particle size distribution. |
| Reaction Conditions / Protocol | DNA-CaCl2-HBS mixing ratio and pH critical; precipitate formation 15-20 min at RT; incubation with cells 4-16 hours at 37 °C, 3-5% CO2. |
| Components / Formulation | 2× HBS buffer (pH 7.08 ± 0.03, 10 mL), 2.5 M CaCl2 solution (1 mL), sterile nuclease-free water (10 mL), detailed protocol with HEK293 cell culture and transfection optimization guide. |
| Storage Conditions | Store 2× HBS and CaCl2 at 2-8 °C; sterile water at room temperature. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 kit (sufficient for ~50 transfections in 100 mm dishes or ~200 transfections in 6-well plates). |
| Product Form | Liquid reagents: 2× HBS (clear), CaCl2 (clear), sterile water. |
| Quality Control | Each lot tested for pH (7.08 ± 0.03), sterility, calcium phosphate precipitate formation quality (fine white precipitate, no large aggregates), and transfection efficiency in HEK293T cells (EGFP >50% positive 48 hr post-transfection). |
| Key Features | Classic calcium phosphate method; extremely low cost per transfection; high efficiency in HEK293 cells; pH-optimized 2× HBS for consistent precipitate; suitable for large-scale protein production; stable transfection compatible; no DNA size limitation. |
| Purity | 2× HBS: molecular biology grade HEPES, NaCl, Na2HPO4; CaCl2 USP grade; water nuclease-free. |
| Concentration | 2× HBS: 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.08; 2.5 M CaCl2. |
| Activity / Unit Definition | Transfection efficiency depends on precipitate quality; fine cloud-like precipitate optimal; large visible aggregates reduce efficiency. |
| Molecular Weight | CaCl2: 110.98 g/mol; HEPES: 238.30 g/mol. |
| Source / Origin | Analytical grade chemicals; nuclease-free water. |
| pH Range / Optimal pH | 2× HBS pH 7.05-7.12 (critical range — pH must be within 7.05-7.12 for optimal precipitate). |
| Shipping Conditions | Ambient or cold pack. |
| Expiration Date / Stability | 12 months at 2-8 °C; verify pH before each use — discard if pH >7.15 (<7.05). |
| Regulatory / Compliance | For research use only; not for therapeutic or diagnostic use. |
| Compatibility | Optimized for HEK293, HEK293T, HEK293FT. Also works with HeLa, CHO-K1, NIH/3T3, COS-7 at lower efficiency. Best results with adherent cells at 50-70% confluence at time of transfection. Serum in medium does not interfere with precipitate formation. |
| Recommended Buffer System | HEPES-buffered saline, pH 7.08. |
| Application Notes / Precautions | The most critical parameter is 2× HBS pH — verify with calibrated pH meter before each use. pH should be adjusted at room temperature (25 °C) as HEPES has significant temperature coefficient (-0.014 pH/°C). Add DNA-CaCl2 mixture DROPWISE to 2× HBS while vortexing — rapid addition causes large aggregates. Use circular plasmid DNA for transient transfection; linearize for stable transfection. Replace culture medium 4-6 hours after transfection to reduce calcium phosphate toxicity. For lentivirus production, reduce DNA amount by 50% to minimize toxicity. |
| Batch-to-Batch Consistency | 2× HBS pH 7.08 ± 0.03; CaCl2 concentration 2.5 ± 0.1 M; precipitate quality verified per lot. |
For research use only, not for clinical use.
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