Designing oligonucleotides and making sure that you have the right parameters for your oligo is an important step in securing results, especially in PCR Primer Design. In order to achieve successful DNA amplification, it’s important to start off with the right primer.
What makes a good primer?
Here are some guidelines for designing your PCR primers:
- Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bondingandhelpwiththestabilityoftheprimer.BemindfulnottohavetoomanyrepeatingG or C bases, as this can cause primer-dimer formation.
- A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.
- Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other. Because the Tm is dependent on the length, it’s important to keep primers on the shorter end. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. 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.
- Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting.
- Try toavoidregionsofsecondarystructureandhaveabalanceddistributionofGC-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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