Skatebro:Primer Design
From OpenWetWare
Tips
- In general, want high GC content, up to 50%
- Insert a double stop codon (taa taa)
- Binding length of 18-22 bps is ideal
- Melt temps. of forward and reverse primers should be within ~2c and ~40-60c is desirable
- analyze the landing sequence and the landing+tail Tm (can gradually increase annealing temp over pcr cycles if necessary)
- Make sure primer can only form short/thermodynamically unstable hairpins
