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Cat. No.: MPPS-10 (for 10T)

Cat. No.: MPPS-50 (for 50T)

Description

Muta-Prem™ Plus Site-Directed Mutagenesis Kit can quickly achieve point mutations, mutations of multiple adjacent codons, deletions, or insertions in plasmids, especially large plasmids (10-35 kb). Site-directed mutagenesis of genes is commonly used to study gene transcription regulation, RNA or protein structure and function, as well as fine-tuning of specific sequences in plasmids.

This kit utilizes the most commonly used technology for gene site-directed mutagenesis. By generating mutated plasmids based on PCR, digesting the template plasmids with DpnI, transforming and culturing the cells, and subsequently performing enzyme digestion or sequencing identification, the desired mutant plasmids can be obtained. The DpnI digestion step takes only 5 minutes, and the total operation time is approximately 2-3 hours to complete site-directed mutagenesis of the gene.

This kit utilizes the latest Extra-long DNA Polymerase, which has improved fidelity, higher sensitivity, and a amplification speed of up to 2 kb/min, enabling rapid and efficient amplification of DNA fragments up to 35 kb in length. This significantly shortens the reaction time required for mutagenesis and further increases the success rate of site-directed mutagenesis. The DpnI enzyme included in the kit has undergone rigorous validation to ensure efficient digestion of methylated template plasmids within 5 minutes, without digesting the unmethylated PCR products.

To use this kit, two primers with complementary sequences of typically 25-45 nucleotides need to be designed, incorporating the desired mutation site. The template for mutagenesis is typically a dam+ E. coli strain such as DH5α, where plasmids are methylated. In vitro PCR amplification of plasmids does not result in methylation. By using the DpnI enzyme, which specifically recognizes methylated sites, the methylated template plasmids can be digested, while the PCR-amplified plasmids containing the desired mutation site are selectively retained. Subsequently, the DpnI-treated products are transformed into competent bacteria, where the nick sites in the mutated plasmids can be repaired by E. coli, resulting in clones containing the desired mutant plasmids.

It has been tested that this kit is capable of performing site-directed mutagenesis PCR amplification on template plasmids up to 14 kb in length.

This kit was tested using the pcDNA3.1-SRCAP plasmid (14566 bp) for site-directed mutagenesis, and the measured mutation rate was close to 100%. However, the actual mutation rate may vary due to factors such as the design of mutation primers, the amount of template used, and the efficiency of transformation.

Precautions

• LB liquid medium and LB agar plates need to be prepared by the user for bacterial culture.
• Primer design and synthesis for site-directed mutagenesis should be done by the user. Reagents for bacterial transformation need to be provided by the user.
• Please read the FAQ section at the back before using this kit.
• This product is intended for scientific research by professionals only and is not to be used for clinical diagnosis or treatment. It should not be used for food or drug purposes and should not be stored in residential areas.
• For your safety and health, please wear laboratory attire and disposable gloves when handling the kit.

Storage

The minimum shelf life is 1 year at -20°C.

Featured Citations

Interested in seeing published research using our Mutagenesis Kits?

NLRP6 potentiates PI3K/AKT signalling by promoting autophagic degradation of p85α to drive tumorigenesis

^{_{Nature Communications    |    28 September 2023    |    DOI: https://doi.org/10.1038/s41467-023-41739-z}}

^{_{p85α mutants were generated using a site-directed mutagenesis kit (SBS Genetech, Beijing, China) according to the manufacturer’s instructions.}}

ZmBSK1 positively regulates BR-induced H2O2 production via NADPH oxidase and functions in oxidative stress tolerance in maize

^{_{Plant Physiology and Biochemistry    |      15 August 2022    |    Doi: https://doi.org/10.1016/j.plaphy.2022.06.011}}

^{_{The mutated ZmCCaMK was obtained using the Site-Directed Mutagenesis Kit (SBS Genetech) followed by the manufacturer's protocol.}}

Rhophilin rho GTPase binding protein 1-antisense RNA 1 (RHPN1-AS1) promotes ovarian carcinogenesis by sponging microRNA-485-5p and releasing DNA topoisomerase II alpha (TOP2A)

^{_{Bioengineered    |      07 Dec 2021    |    Doi: https://doi.org/10.1080/21655979.2021.2002494}}

^{_{The site-directed mutagenesis kit (SBS Genetech, China) was used to mutate the WT binding sequence of RHPN1-AS1 or TOP2A 3ʹ-UTR, and the produced mutant sequence was also inserted into psiCHECK2 vectors, which were named RHPN1-AS1 Mut1, RHPN1-AS1 Mut2, RHPN1-AS1 co-Mut, and TOP2A Mut vectors.}}

Thr420 and Ser454 of ZmCCaMK play a crucial role in brassinosteroid-induced antioxidant defense in maize

^{_{Biochemical and Biophysical Research Communications Supports open access    |      7 May 2020    |    Doi: https://doi.org/10.1016/j.bbrc.2020.02.078}}

^{_{The Site-Directed Mutagenesis Kit (SBS Genetech, China) was used to obtain the site-directed ZmCCaMK according to the manufacturer’s instructions.}}

Long non-coding RNA LOC554202 promotes acquired gefitinib resistance in non-small cell lung cancer through upregulating miR-31 expression

^{_{Journal of Cancer    |     15 Oct 2019    |    Doi: https://doi.org/10.7150%2Fjca.35097}}

^{_{The mutation of miR-31 binding sites in 3'-UTRs of RASA1 (UCUUGCC was mutated to UGUUCGG) or FIH-1 (UCUUGCC was mutated to UGUUCGG) was performed by a Muta Direct Site-directed Mutagenesis kit (SDM-15; Beijing SBS Genetech Co, Ltd, Beijing, China).}}

Long Non-coding RNA H19 Inhibits Adipocyte Differentiation of Bone Marrow Mesenchymal Stem Cells through Epigenetic Modulation of Histone Deacetylases

^{_{Scientific Reports    |     28 June 2016    |    DOI: https://doi.org/10.1038/srep28897}}

^{_{Site-directed mutagenesis of the H19 sequences was performed using the Site-Directed Mutagenesis Kit (SBS Genetech, Beijing, China).}}