A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing. These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal. Analysis of primer sequences
Good primer design is essential for a successful PCR reaction. There are many factors to take into account when designing the optimal primers for your gene of interest. Here are some tips to consider when designing primers.
In general, a length of 18–30 nucleotides for primers is good.
Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other.
If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
Typically, 3 to 4 nucleotides are added 5’ of the restriction enzyme site in the primer to allow for efficient cutting.
Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
If you are using the primers for a PCR reaction to be used in Invitrogen™ TOPO™ cloning, the primers should not have a phosphate modification.
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