- ALWAYS make sure primer are specified in 5’ to 3’ orientation.
- Primer length for PCR and SEQUENCING should be in between 18-25 bp.
- GC content should be in between 40-60%.
- 3’ end should end with G or C (GC clamp) with at least 2 G or C in last 5 nucleotides.
- Avoid 4 or more of one base together (AAAAC, TGCCCC…), as this can lead to mispriming.
- Avoid intra and inter primer homology i.e. they should not be complementary to each other which may lead to formation of primer dimers. Complementary within the primer can lead to hairpin formation, avoid this.
- For better amplification the difference in Tm (melting temperature) between forward and reverse primer should be <5.
- Tm between 58 degree Celsius to 65 degree Celsius is preferable for better amplification.
Some websites for primer design