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| Product Name | Site-Directed Mutagenesis Kit |
| Catalog No. | NATR-HMM-0132 |
| Description | A complete kit for introducing point mutations, insertions, and deletions into plasmid DNA using a high-fidelity DNA polymerase and DpnI restriction enzyme-based template removal strategy. The entire mutagenesis reaction is performed in a single tube, followed by direct transformation of the amplified product. |
| Intended Use | Introduction of site-specific mutations (substitutions, deletions, insertions) into plasmid vectors for protein structure-function studies, promoter analysis, epitope tagging, restriction site addition or removal, and generation of mutant cDNA libraries. |
| Principle / Technology | Complementary mutagenic primers carrying the desired mutation are extended around the entire plasmid template by a high-fidelity DNA polymerase in a linear amplification reaction. The parental (non-mutated) methylated plasmid DNA template is selectively digested by DpnI endonuclease, which cleaves only Dam-methylated DNA at GATC sequences. The nicked, non-methylated mutant plasmid is then transformed into competent E. coli cells, which repair the nicks and replicate the mutant plasmid. |
| Detection Method | Sanger sequencing of plasmid miniprep DNA from transformed colonies to verify desired mutation and confirm absence of secondary mutations; restriction digestion when the mutation creates or destroys a restriction site; colony PCR for preliminary screening |
| Sample Type | Dam-methylated plasmid DNA (from E. coli strains such as DH5α, XL1-Blue, JM109, TOP10) carrying the target gene; compatible with plasmids up to 15 kb; not suitable for non-methylated plasmids from dam– E. coli strains |
| Performance Range / Specifications | Mutation efficiency: >80% of colonies contain the desired mutation; plasmid template size: up to 12–15 kb (standard); mutagenic primer length: 25–45 nucleotides; mutation types: single or multiple base substitutions, deletions up to 3 kb, insertions up to 12 amino acids (with long primers), and sequential mutagenesis of multiple sites |
| Sensitivity / LOD | Reliable introduction of single base-pair mutations; ability to introduce mutations at multiple sites by sequential rounds of mutagenesis |
| Specificity | DpnI digestion removes >99.9% of parental methylated template, reducing false-positive background; colony screening typically confirms >80% mutant rate for single-site mutations; Sanger sequencing confirms correct mutation without unwanted secondary mutations |
| Reaction Conditions / Protocol | Design complementary mutagenic primers containing the desired mutation flanked by 10–15 bp of correct sequence on each side; set up 50 μL PCR: template DNA (5–50 ng), forward and reverse mutagenic primers (125 ng each), dNTPs, high-fidelity polymerase, buffer; thermal cycling: 95°C 30 sec, 12–18 cycles of (95°C 30 sec, 55°C 1 min, 68°C 2 min/kb of plasmid), 68°C 5 min final extension; add 1 μL DpnI to PCR product, incubate 37°C 1 hour; transform 2–5 μL into competent cells |
| Components / Formulation | High-fidelity DNA polymerase blend, 10× reaction buffer with MgSO4, dNTP mix, DpnI restriction enzyme (10 U/μL), control plasmid and primer set, nuclease-free water; competent cells optional or sold separately |
| Storage Conditions | All reagents at –20°C (polymerase, dNTPs, DpnI); DpnI should be kept on ice during use; buffer may be stored at –20°C or room temperature |
| Shelf Life | 12 months at –20°C from date of manufacture |
| Package Specifications | 30 reactions and 100 reactions |
| Product Form | Liquid enzymes dNTPs, and control materials |
| Quality Control | Each lot tested: control point mutation achieves >80% mutant colonies; DpnI digestion efficiency >99.9% (no colonies in mock-transformed DpnI-only digestion); polymerase fidelity: <1 secondary mutation per 6 kb of plasmid DNA in 10 sequenced clones; control plasmids verified by sequencing |
| Key Features | Single-tube mutagenesis with no subcloning or single-stranded DNA preparation; DpnI template elimination ensures low background; high-fidelity polymerase minimizes unwanted secondary mutations; support for large plasmids up to 15 kb; validated primers included as positive control for new users |
For research use only, not for clinical use.
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