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 TOPO cloning, the primers should not have a phosphate modification.
Designing & ordering primers with Vector NTI Advance software
Designing and ordering primers takes about 60 seconds when you use Vector NTI Advance™ Sequence Analysis Software:
- Select the region to amplify.
- Access the primer design menu and select “amplify selection”.
- Add 5' and 3' restriction sites, if required, to the sense and antisense primer.
- Add additional bases, if required, to the sense and antisense primer.
- Inspect the left pane for thermodynamic parameters such as Tm, length, and GC content.
- Select the product, and click “order”.
- Add a researcher name, any desired 5' or 3' modifications, or change the purity.
- Click “add to cart” and begin the checkout process.
OligoPerfect™ Designer is a free, simple, and efficient Web-based primer design tool that works with a DNA template sequence you upload. Like Vector NTI Advance software, OligoPerfect™ Designer is seamlessly connected to our online ordering system, so you never have to cut and paste sequences.
Primer Designer Tool
The primer designer tool lets you quickly search for, configure and order primers for the Sanger confirmation step of your NGS workflow. The database consists of ~650,000 pre-designed primer pairs for re-sequencing the human exome and human mitochondrial genome.
- Our primers are free of SNPs and primer-dimers, highly target-specific, and used under universal PCR conditions
- Full primer coverage for Ion AmpliSeq™ Exome Panel and Ion AmpliSeq™ Cancer Hotspot Panel v.2 Sanger confirmation workflow
- Flexible primer configuration to meet your research needs: primers can be ordered unmodified, M13-tailed, HPLC-purified, or desalted
- All the primers have been checked by mass spectrometry and have passed stringent bioinformatics metrics. Lab bench validation test have shown >95% success rate.
For Research Use Only. Not for use in diagnostic procedures.