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| Product Name | TA Cloning Vector Kit (pGEM-T Easy System) |
| Catalog No. | NATR-HMM-0131 |
| Description | A convenient linearized plasmid vector system with single 3ʹ-terminal thymidine overhangs at both ends for direct cloning of PCR products generated by Taq and other non-proofreading DNA polymerases that add 3ʹ-A overhangs. The vector contains convenient restriction sites flanking the insertion site for easy excise of cloned inserts. |
| Intended Use | Direct cloning of Taq-amplified PCR products for sequencing, transcription, expression analysis, and downstream subcloning. The system is designed for routine cloning of PCR fragments where proofreading polymerase fidelity is not required for the initial amplification. |
| Principle / Technology | Taq DNA polymerase and other non-proofreading polymerases add a single deoxyadenosine to the 3ʹ ends of PCR products. The pGEM-T Easy vector is supplied linearized with single 3ʹ-T overhangs that are complementary to the PCR product A-overhangs. T4 DNA ligase catalyzes the linkage of the complementary T/A ends, greatly increasing the efficiency of direct PCR product insertion compared to blunt-end cloning of unmodified fragments. |
| Detection Method | Blue-white colony screening on LB agar plates containing ampicillin, IPTG, and X-Gal; white colonies indicate recombinant plasmids with interrupted lacZ α-peptide coding sequence; colony PCR and restriction digestion for insert verification; Sanger sequencing with T7 and SP6 promoter primers for insert confirmation and orientation |
| Sample Type | PCR products amplified with Taq or other non-proofreading DNA polymerases (GoTaq, AmpliTaq, DreamTaq, etc.); PCR products with 3ʹ-A overhangs; compatible with fragments 100 bp to 8 kb |
| Performance Range / Specifications | Insert size range: 100 bp to >5 kb (standard), >8 kb with adjusted conditions; cloning efficiency: 1 × 10^6 to 1 × 10^8 CFU/μg vector (competent cell strain dependent); typical white colony percentage: 60–90% (background blue colonies from vector self-ligation or incomplete digestion) |
| Sensitivity / LOD | Successful cloning from as little as 10 ng of purified PCR product; background blue colonies typically <20% with proper vector-to-insert ratios |
| Specificity | Blue-white screening specifically identifies recombinant clones; lacZ-based screening is highly specific for insert-containing plasmids; minimal false-positive white colonies (typically <5% due to frame-shift or small deletion mutations in lacZα) |
| Reaction Conditions / Protocol | Ligation: mix 50 ng vector, insert DNA (3:1 molar ratio), 3 U T4 DNA ligase, 1× rapid ligation buffer; incubate 1 hour at room temperature or overnight at 4°C; transformation: add 2 μL ligation to 50 μL competent cells, incubate on ice 20 min, heat shock 42°C 45–50 sec, ice 2 min, add 950 μL SOC, incubate 37°C 1–1.5 hours, plate on LB/ampicillin/IPTG/X-Gal plates, incubate 37°C overnight |
| Components / Formulation | pGEM-T Easy vector (50 ng/μL, linearized with 3ʹ-T overhangs, in TE buffer), T4 DNA ligase, 2× rapid ligation buffer, control insert DNA, nuclease-free water, competent E. coli cells (optional, sold separately or pre-packaged in complete kit) |
| Storage Conditions | Vector: –20°C (stable 24 months); ligase and buffer: –20°C; control insert: –20°C; avoid repeated freeze-thaw of vector DNA; protect from excessive light exposure |
| Shelf Life | 24 months at –20°C from date of manufacture; competent cells (if included) require –80°C storage |
| Package Specifications | 20 reactions; complete version includes competent JM109 or DH5α cells |
| Product Form | Linearized plasmid DNA vector (liquid); T4 DNA ligase (liquid); optional chemically competent cells (frozen suspension) |
| Quality Control | Each lot validated: cloning of 542 bp control insert, >1 × 10^8 CFU/μg efficiency with provided competent cells; >80% white colonies; sequencing confirms inserts in both orientations; functional T7/SP6 promoter sequencing verified; no detectable exonuclease activity in vector preparation |
| Key Features | Pre-linearized T-overhang vector saves restriction digestion steps; blue-white colony screening simplifies clone identification; multiple restriction enzyme sites (EcoRI, NotI, BstZI) for convenient insert excision; T7 and SP6 RNA polymerase promoters flanking the insert for in vitro transcription; lacZα complementation in a wide variety of common E. coli host strains |
For research use only, not for clinical use.
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