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PCR cloning primer design method

Update time : 2020-08-17

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PCR cloning primer design method

PCR cloning is a molecular biology technology that amplifies and expands specific DNA fragments, and is widely used in molecular biology and related fields. 

Among them, PCR cloning primer is an important factor in the process of PCR expansion reaction. Therefore, before the PCR cloning experiment is officially started, it is a necessary skill to master the correct PCR cloning primer design method.

PCR cloning primer design process:

Obtain PCR extension fragment sequence: In the first case, expand based on known fragments, and you need to design PCR expansion primers through NCBI search for gene sequences; in the other case, expand unknown gene fragments, you need to search for conservative sequences of related species, based on conservative sequences DNA or RNA as template to design primers.

For example, Prime Premier 5.0 primer design software is powerful and easy to use. It can compare and comprehensively evaluate the design of primers. It is one of the most commonly used software for primer design.

PCR extension test verification: PCR amplification can be carried out after primer synthesis is completed, and the correctness of PCR cloning primers can be predicted based on the length of the amplified product by gel electrophoresis.

Precautions for PCR cloning primer design:

The primer length is basically maintained between 18-24bp. If the length of the primer sequence is too short, it may expand and affect the expansion rate of PCR cloning; if the length of the primer is too long, the primer reduction cannot be increased, but the too long sequence will cause mismatches and reduce the expansion rate of PCR cloning.

Therefore, the appropriate Tm value is 55-80°C, and the initiation temperature difference between the upstream and downstream primers should be within 10°C. 

Therefore, the GC content of the extension will affect the melting temperature of the oligonucleotide during the PCR cloning process, that is, the Tm value. 

Generally, the GC content of the extension is between 40%-60%, and the GC content difference between the upstream and downstream primers is within 20%, which can increase the primer content.

If this happens, mismatches may occur during the PCR process, which will affect the accuracy of PCR amplification.

Prevent the single strand of the amplification primer from forming a secondary structure during the PCR cloning process and inhibit the PCR expansion process.

Generalbiosystems' gene synthesis platform uses patented cloning technology to help customers clone the target gene at any designated position on the desired vector without relying on vector restriction sites to meet various cloning design plans for customers.