Controlled Error-prone PCR Kit
Error front PCR utilizes the characteristic of Taq DNA polymerase that does not have 3 '→ 5' proofreading function. Under certain conditions (such as different dNTP concentrations, Mg concentrations, and MnCl2), it can introduce random mutations with a high probability. If there is an appropriate selection method, the required mutants can be selected from the constructed mutation library. This product is an improvement on our company's error prone PCR kit.
Ready to use, it is very simple and convenient, especially in the fields of biopharmaceutical and enzymatic research, demonstrating its superiority.
The formula has been carefully optimized, and the experimental personnel can control the mutation rate within a certain range. The mutation rate range for one round of PCR is 2-8 mutations/1000bp, and higher mutation rates can be achieved through multiple rounds of PCR.
It can be used for sequential error front PCR, where the product of the previous PCR can be used as a template for the next PCR, resulting in the accumulation of important beneficial mutations with each small mutation obtained.
The DNA fragments that can be amplified are longer, reaching 2kb. For products above 2kb, segmented amplification is recommended.
This reagent kit is sufficient for 100 times 30 μ L system error prone PCR.
This product can only be used for scientific research.
Component specifications
Adjustable error prone PCR specific Taq DNA polymerase (5U/ μ L) 50 μ L
five × Adjustable and error prone PCR Mix (including dNTP) 600 μ L
Tunable error prone PCR dedicated MnCl2 300 μ L
Adjustable and error prone PCR dedicated dGTP 300 μ L
ten × Adjustable error prone PCR with dNTP 300 added μ L
Storage: -20 ℃, valid for one year.
Primers, DNA templates, and conventional PCR kits.
Dilute the primer to 10 μ M. Primers are also a key factor in determining the success or failure of error prone PCR. When designing primers, it is necessary to ensure that their Tm is at 70 ℃, their length is between 25-30 nt, their GC content is between 45% and 60%, their 3-terminal GC content is low, and there is no hairpin structure.
Prepare error prone DNA templates (conventional PCR products) by amplifying with self prepared error prone PCR primers and self prepared conventional PCR reagents, and accurately determine the concentration through gel recovery. Note: The template for error prone PCR must use conventional PCR products that are recycled using gel, as gel recycling can remove non specific amplification fragments that are not visible on electrophoresis. Otherwise, these fragments, which are also amplified using error prone PCR primers, will also be amplified during error prone PCR amplification, resulting in competitive inhibition and reducing the amplification efficiency of the target molecule.
Dilute the recovered DNA fragments (templates) to 1ng/ μ L. 10ng/ μ L and 100ng/ μ Three concentrations of L.
Note: Template usage is the most important factor affecting mutation rate. The template DNA is non mutated DNA, and the amplified DNA is mutated DNA. The final DNA amount in the error prone PCR system is basically fixed. Therefore, the larger the template DNA usage, the better the comparison of mutated DNA in the final product