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Design primers for Gibson cloning

  1. Encode Gibson homology overlap in only one of the primers (typically in the region that you expect to remain constant in future cloning). This will let you re-use primers between different experiments.
  2. Keep homlogy overlap to between 20-24nt. Adjust length to have at least 52C annealing temperature on SnapGene.
  3. Keep PCR primer binding regions to 17-22nt so that the annealing region has a Tm of >50C (ideally 60C) and a GC content of 35-65%.
  4. Keep all primers to <=60nt since longer primers are both more expensive and have higher error rate.