Skatebro:Primer Design: Difference between revisions
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*Insert a double stop codon (taa taa) | *Insert a double stop codon (taa taa) | ||
*Binding length of 18-22 bps is ideal | *Binding length of 18-22 bps is ideal | ||
*Melt temps. of forward and reverse primers should be within ~2c and ~ | *Melt temps. of forward and reverse primers should be within ~2c and ~50-60c is desirable | ||
**analyze the landing sequence and the landing+tail Tm (can gradually increase annealing temp over pcr cycles if necessary) | **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 | *Make sure primer can only form short/thermodynamically unstable hairpins | ||
Revision as of 13:49, 7 February 2008
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 ~50-60c 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
