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| Product Name | Multi Site-Directed Mutagenesis Kit |
| Catalog No. | RESK-0071 |
| Description | The Multi Site-Directed Mutagenesis Kit is a rapid mutagenesis system built on fast homologous recombination cloning technology. With this kit, site-directed mutations can be introduced simultaneously at three to five non-contiguous sites (spaced more than 50 bp apart) on a target plasmid in a single reaction. The kit consists of two modules: a high-fidelity Pfu DNA polymerase amplification module and a rapid homologous recombination cloning module. The ultra-high fidelity of the Pfu polymerase (83-fold higher fidelity) greatly reduces the chance of introducing unwanted mutations during amplification, and its outstanding long-fragment amplification capability supports plasmids up to 20 kb. The homologous recombination system replaces the traditional annealing-and-circularization reaction, making primer design more flexible; amplification proceeds exponentially, greatly reducing template input and favoring complete degradation of the original methylated template. The recombinase in this kit is specifically optimized for multi-base site-directed mutagenesis, and when the amplification product is specific, the DpnI-digested product can be used directly in the recombination reaction without DNA purification. |
| Intended Use | Simultaneous introduction of site-directed mutations at 3-5 non-contiguous sites (more than 50 bp apart) on a target plasmid. |
| Principle / Technology | Based on rapid homologous recombination cloning technology: the target plasmid is amplified with a high-fidelity Pfu DNA polymerase using primers carrying the desired mutations, the methylated parental template is removed by DpnI digestion, and the linear amplified product is circularized by an optimized multi-fragment recombinase through an efficient homologous recombination reaction, followed directly by transformation. |
| Sample Type | Methylated plasmid DNA (propagated in methylation-competent hosts such as DH5α, TOP10, or JM109), up to 20 kb in length. |
| Performance Range / Specifications | Introduces mutations at 3-5 non-contiguous sites in one step Polymerase fidelity: 83-fold higher than conventional Taq Applicable to plasmids up to 20 kb Annealing temperature: 60-72°C Extension time: 30-60 s/kb Reaction time: 20-50 min Non-contiguous sites are defined as mutation sites spaced more than 50 bp apart |
| Specificity | The recombinase multi-fragment-optimized recombinase is specifically optimized for site-directed mutagenesis of multiple bases. |
| Reaction Conditions / Protocol | 1. Amplify the target plasmid by PCR with the high-fidelity Pfu DNA polymerase using mutation-carrying primers. 2. Digest the methylated parental template with DpnI. 3. Perform the homologous recombination circularization reaction (the DpnI-digested product can be used directly without purification if amplification is specific). 4. Transform the recombination product directly into competent cells. |
| Components / Formulation | High-fidelity Pfu DNA polymerase amplification module Rapid homologous recombination cloning module (containing the optimized recombinase multi-fragment-optimized recombinase) |
| Storage Conditions | Store at -20°C. |
| Shelf Life | 1 year. |
| Package Specifications | 10 T |
| Product Form | Kit |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Valid for 1 year when stored at -20°C. |
| Application Notes / Precautions | The multi-site kit introduces mutations at 3-5 non-contiguous sites in one step; for 1 or 2 non-contiguous sites, a standard site-directed mutagenesis kit is sufficient. Three consecutive mutated bases count as a single site; sites more than 50 bp apart are considered non-contiguous. Use a methylated plasmid as PCR template so that DpnI can digest the parental template and prevent false positives; propagate the original plasmid in methylation-competent hosts (e.g., DH5α, TOP10, JM109). Use freshly prepared plasmid as template, as prolonged storage and repeated freeze-thaw cycles may cause template breakage, nicking, or degradation. For inverse PCR amplification of the plasmid, design primers so that the 5' ends of the forward and reverse primers contain at least 20 bp of reverse-complementary region, with each non-complementary region at least 20 bp long; choose complementary regions without repetitive sequences and with a GC content of 40%-60%. |
For research use only, not for clinical use.
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