PCR-based random mutagenesis is an important tool for elucidating structure-function relationships and improving protein properties. Error-prone PCR is a technique to introduce random sequence mutations into target genes by utilizing Taq DNA Polymerase, which does not have a 3′ → 5′ proofreading function, in a specific PCR reaction system. The kit is optimized by adjusting the amount of Mn2+ and dGTP concentrations using Mutation DNA Polymerase introduces varying numbers of base mutations into the target gene, increasing the likelihood of generating amino acid sequence mutations at the protein level. The amplification products of random mutations are cloned into expression vectors to construct gene libraries, which are then transferred into expression hosts to screen the proteins for activity, enabling researchers to screen for beneficial mutations in the absence of structural information or when it is difficult to predict the beneficial mutations from the protein structure.
| individual parts making up a compound | norm |
| Mutation DNA Polymerase (1 U/μl) | 50 μl |
| 5X Mutation Buffer | 500 μl |
| 50X dNTP Mix (dGTP free) | 50 μl |
| dNTP Mix (10 mM each)* | 50 μl |
| dGTP Solution (2 mM) | 250 μl |
| MnSO4 Solution (8 mM) | 200 μl |
*: Standard dNTP mix with 10 mM of dATP, dCTP, dGTP and dTTP.
1. Controlled random mutagenesis, whereby reaction conditions can be varied to achieve the desired level of random mutagenesis.
2. good specificity, the product is added with monoclonal antibody that can inhibit the activity of DNA polymerase, so that hot start reaction can be carried out.
3. High amplification performance, with DNA fragments up to 4.1 kb in length under all reaction conditions.
4. Mutational diversity, which can produce point mutations for transitions and transversions.
(Note: Conversion mutations include purine → purine and pyrimidine → pyrimidine changes, and subversion mutations are purine ↔ pyrimidine changes.)
5. The amplified PCR product has an A base at the 3′ end and can be cloned directly into a T vector.
Storage temperature: -20℃
Transportation temperature: dry ice transportation or -20℃ ice bag transportation
