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| Product Name | LR Clonase II Enzyme Mix for Gateway Cloning |
| Catalog No. | NATR-HMM-0142 |
| Description | A proprietary enzyme mixture containing the integrase (Int) and integration host factor (IHF) proteins from bacteriophage lambda for the site-specific recombination-mediated transfer of DNA sequences between Gateway entry and destination vectors in vitro. |
| Intended Use | Transfer of gene-coding sequences from Gateway entry clones to destination vectors for expression in bacterial, mammalian, insect, and yeast systems, enabling rapid construction of expression clones without traditional restriction enzyme and ligase reactions. |
| Principle / Technology | The LR recombination reaction catalyzes the in vitro recombination between attL sites (flanking the gene in entry clones) and attR sites (flanking the ccdB counterselection cassette in destination vectors). Lambda integrase (Int) catalyzes the strand exchange, while integration host factor (IHF) facilitates proper att site synapsis. The resulting expression clone contains the gene flanked by attB sites, while the by-product plasmid carries the ccdB gene and confers toxic effects in non-resistant E. coli strains, providing selection for the desired recombinant. |
| Detection Method | Transformation of recombination products into E. coli; antibiotic selection (ampicillin or carbenicillin for expression vector, plus ccdB counterselection against by-products); colony PCR, restriction digestion, and sequencing for verification of correct insert transfer; protein expression analysis for functional confirmation |
| Sample Type | Purified entry clone plasmid DNA containing the gene of interest flanked by attL sites; compatible with any Gateway destination vector carrying attR sites and selectable markers; starting material: 50–150 ng each of entry clone and destination vector |
| Performance Range / Specifications | Recombination efficiency: >5 × 10^7 CFU/μg plasmid DNA (with DH5α competent cells); correct transfer of DNA inserts up to 11 kb; >99% of colonies carry the desired expression clone with correct insert; reaction time: 1 hour at 25°C (standard), overnight at 25°C for maximum efficiency |
| Sensitivity / LOD | Reliable transfer of inserts from picogram quantities of entry clone; background <1% (parental destination vector colonies from incomplete ccdB selection); successful with inserts as large as 11 kb verified by sequencing |
| Specificity | Site-specific recombination ensures precise, scarless transfer of the DNA segment between att sites; attB1 and attB2 sites in the expression clone are 25 bp and maintain reading frame consistency; ccdB counterselection eliminates >99.9% of non-recombinant destination vector background |
| Reaction Conditions / Protocol | Set up 10 μL reaction at room temperature: entry clone (50–150 ng), destination vector (150 ng), TE buffer to 8 μL, add 2 μL LR Clonase II enzyme mix, mix by pipetting, incubate at 25°C for 1 hour (or overnight), add 1 μL Proteinase K solution, incubate at 37°C for 10 minutes to terminate reaction, transform 2 μL into competent E. coli (DH5α, TOP10, or equivalent), plate on selective medium |
| Components / Formulation | LR Clonase II enzyme mix (integrase, integration host factor, proprietary buffer), Proteinase K solution (2 μg/μL, for reaction termination), positive control (pENTR-Gus entry clone and pDEST-15 destination vector), nuclease-free water |
| Storage Conditions | Enzyme mix: –80°C or –20°C; stable at –20°C for up to 6 months after initial thaw; avoid repeated freeze-thaw (>5 cycles); proteinase K: –20°C; control plasmids: –20°C; all components should remain on ice during use |
| Shelf Life | 12 months at –20°C from date of manufacture; 6 months at –20°C after first thaw |
| Package Specifications | 20 reactions × 10 μL and 100 reactions × 10 μL |
| Product Form | Liquid enzyme mixture in storage buffer (contains glycerol); control plasmids provided separately |
| Quality Control | Each lot tested: >5 × 10^7 CFU/μg efficiency with control entry clone and destination vector; >99% correct recombinants by colony PCR (≥20 colonies); sequencing verifies precise attB site formation with correct reading frame; negative control (no enzyme) shows zero colonies; functional expression of control protein in the appropriate host system verified |
| Key Features | One-hour reaction time for standard cloning; >99% recombination accuracy with ccdB counterselection; scarless and in-frame gene transfer preserving codon reading frame; compatible with >50 commercially available destination vectors for multiple expression systems; no restriction digestion, gel purification, or ligation steps |
For research use only, not for clinical use.
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