The Methods of PCR Cloning

PCR Cloning is a technique used to amplify a specific region of DNA strand, and is used in almost every molecular biology lab in the world. PCR can be used on almost any DNA region, provided that suitable primers can be made. Synbio Tech provides one-stop PCR cloning services, including primer design.

PCR Primer Design Procedure

  • Acquisition of DNA sequence: For amplification of known DNA sequences, tried-and-tested primers can be found on the NCBI website. For unknown DNA sequences containing conserved sequence(s) from related species, primers should be designed according to the DNA or RNA of the conserved sequence(s).
  • PCR primer design: Many commercial software products and online tools are used to design primers. Primer Premier 5.0, the most popular primer design software, is both powerful and convenient to use. It can also contrast and comprehensively assess the best choice of primer according to their specificity.
  • Validation of PCR amplification: Once primer synthesis finishes, the accuracy of the primer can be predicted after gel electrophoresis of PCR product.

Notes About PCR Primer Design

  • The length of primer should be around 18-24bp. If the primer is too short, the primer specificity will be too low; if the primer is too long, it may cause base pair mismatches and may reduce the PCR amplification efficiency.
  • The GC% of the primer will affect the denaturation temperature (Tm). Tm should be around 55-80℃ and the annealing temperature difference between the upstream and downstream primers should be within 10℃. Usually, the GC% of primer should be between 40%-60% ,and the GC% difference between the upstream and downstream primers should be within 20% of each other in order to enhance primer specificity.
  • Repetitive structures or high similarity with the template sequence should be avoided as both may lead to possible base pair mismatches.
  • Secondary structure of primers may inhibit the PCR reaction, and should be avoided.

Synbio Technologies provide one-stop PCR cloning services, including primer design. Our Syno® 2.0 platform can clone the target gene to any specific point on a provided vector without relying on restriction enzyme sites, to best satisfy the cloning requirements of each client.