Primer Design Tips & Tools 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-pri