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This help section assumes that you have already read about Gibson Assembly here too.

Primer Design

There are several criteria to meet when designing primers for Gibson assembly. These fall into two categories - melting temperature and priming.

Melting Temperature Self/mispriming
  • The whole primer must have a melting temperature of much more than 50°C (the temperature at which Gibson Assembly is performed)
  • The annealing temperature for the pair of primers for DNA should be about the same (for conveniences sake)
  • The primer must not self-anneal
  • The annealing portion of the primer must be specific to only the end of the target DNA
  • The flappy end of the primer must be specific only to the end of the adjacent DNA

All of these can factors can be influeced by adjusting the length of each half (annealing and flappy) of the primer.

The general method used by the Gibthon Construct Designer is as follows

  1. Set a target temperature for the primers:
    • Tm for annealing portion: 50°C
    • Tm for whole primer: 60°C
  2. Set a minimum length for the primers:
    • 20 bp for each half
  3. Generate sequences for the primers
  4. For each primer:
    1. Increase the length of the annealing portion until Tmanneal ≥ 50°C
    2. Increase the length of the flappy end until Tmwhole ≥ 60°C
    3. Check for mispriming/selfpriming, specifically of the 3' end, and warn the user
    4. The user may then choose to adjust the length and sacrifice Tm to avoid mispriming

Done by hand, this technique works but can take several hours per construct. The GCD can do it in about 10 seconds!

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