You can perform a BLAST search to see if the 3′ portion of your primer might hybridize to unintended sites on your template.Complementarity between primer pairs to avoid primer dimers.Complementarity within your primers to prevent hairpin structures.The last five nucleotides at the 3′ end should contain no more than two guanines or cytosines. Runs of identical nucleotides in the 3′ portion of the primer.All remaining cycles should be based on the full-primer T m. NOTE: During PCR amplification, the first-cycle T m should be based on just this 3′ portion of the primer. If the T m is too low, increase the length of the gene-specific portion to reach a T m between 58–65☌. The primer T m is calculated from the 3′ (gene-specific) end of the primer, NOT the entire primer.(See Tip 4 for details on calculating the T m.) If the difference between the T ms for your forward and reverse primers is >4☌, you are not as likely to get efficient amplification. Aim for melting temperatures (T ms) between 58–65☌.If you are using In-Fusion Cloning, this overlap can be shortened to 15 bases for single-insert cloning and 20 bases for multiple-insert cloning. Most seamless cloning protocols, including the Gibson method, require the 5′ end of the primer to contain 25 bases that are homologous to one end of the DNA fragment to which it will be joined (i.e., the vector or another insert).This sequence should be 18–25 bases long and should ideally have a GC content between 40–60%. The 3′ end of the primer should contain sequence that is specific to the target gene you are amplifying.Interested in trying seamless PCR cloning but don't know where to start? Or maybe you just need a quick refresher? Here's a list of top tips to keep in mind when designing your primers for seamless cloning, including some information specific to In-Fusion Cloning.
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