Mike Barnkob:Protocols/Cloning/Primer Design
General notes and guidelines on primer design.
- Primer length: Between 18-25 bp
- Melting temperature, Tm: 52-58°C
- GC Content: Between 40-60%.
- GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.
- Secondary structures: Check for hairpins, self-dimers and cross-dimers at OligoAnalyzer
Hair-pins: Larger negative value for ΔG indicates stable, undesirable hairpins.
Self-dimers: A 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated generally.
Cross-dimers: Interaction between sense and antisense primers, where they are homologous. Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.
- Repeats: Avoid di-nucleotides such as ATATAT. Maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-nucleotides.
- Cross homology: Test primers to avoid amplifying other genes in the mixture by blasting at NCBI Blast
- Dilute the primers into a stock solution, usually 100 pmol/uL.
- Create working-stock by further diluting to 5 pmol/uL.
- Store in the dark
- Stable at 4°C in dH20 for up to 60 weeks
- Can be frozen at -20°C