The following recommendations are provided to help optimize primer selection:
- Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA.
- Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs should be avoided.
- Keep the G-C content in the range of 50-55%.
- For primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm>55oC.
- Avoid primers that have secondary structure or that can hybridize to form dimers.
For each reaction requested, please provide 3 uL of primer at a concentration of 5 pmol/uL (uM).