- Home
- IVD
- By Technology Types
- By Diseases Types
- By Product Types
- Research
- Resource
- Distributors
- Company
| Product Name | Site-Directed Mutagenesis Kit |
| Catalog No. | RESK-0072 |
| Description | The Site-Directed Mutagenesis Kit is a rapid mutagenesis system based on fast homologous recombination cloning technology. With this kit, site-directed mutagenesis is completed simply by DpnI digestion of the target plasmid amplification product, homologous recombination-based circularization, and direct transformation. 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) significantly reduces the possibility of introducing new mutations during amplification, and its excellent long-fragment amplification capability is broadly applicable to any plasmid up to 20 kb. The homologous recombination system replaces the traditional annealing-and-circularization reaction, allowing more flexible primer design and exponential rather than linear amplification, which greatly reduces template input and facilitates complete degradation of the original methylated template. Because the recombination technology efficiently joins two PCR products seamlessly, the kit can also mutate two non-contiguous sites on a target plasmid simultaneously in a single amplification. The optimized recombinase is dedicated to single-base and non-contiguous dual-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 | Site-directed mutagenesis of a target plasmid at a single site (single base or consecutive multiple bases) or at two non-contiguous sites (more than 50 bp apart). |
| Principle / Technology | Based on homologous recombination cloning technology: the target plasmid is amplified by PCR with a high-fidelity Pfu DNA polymerase using mutation-carrying primers, the methylated parental template is removed by DpnI digestion, and the amplified product is circularized by an optimized recombinase (optimized recombinase) via 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 | Suitable for single-base, consecutive multi-base, or non-contiguous dual-base (sites >50 bp apart) mutagenesis 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 min |
| Specificity | The optimized recombinase is specifically dedicated to single-base and non-contiguous dual-base site-directed mutagenesis. |
| 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) |
| 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 | This kit suits 1 or 2 non-contiguous sites; three consecutive mutated bases count as one site, and sites more than 50 bp apart are considered non-contiguous. For 3-5 non-contiguous sites, use the multi-site mutagenesis kit. 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) and use freshly prepared plasmid. Use as little plasmid template as possible (recommended <1 ng) without compromising amplification, to avoid incomplete DpnI digestion. Gel recovery of the amplification product is recommended after DpnI treatment, as incompletely digested plasmid or non-specific bands can impair the subsequent recombination reaction. For inverse amplification, design primers with at least 20 bp of reverse-complementary region at the 5' ends and at least 20 bp in each non-complementary region; avoid repetitive sequences and keep GC content within 40%-60%. Suitable for plasmids smaller than 20 kb; larger plasmids increase off-target mutation rates, make PCR more difficult, and lower recombination efficiency. |
For research use only, not for clinical use.
|
There is no product in your cart. |
Copyright © 2026 Alta DiagnoTech. All rights reserved.
