Gibson Assembly


  1. Assembly fragments were designed to have 35-40bp overhanging regions of complementary in the primers
  2. PCR fragments were generated using a KAPA HiFi polymerase reaction. Fragments were verified on an agarose gel and treated with DpnI restriction enzyme
  3. The following reaction was set up: 0.5 pmols of linear fragments, at least two fold excess of inserts and 75ng of vector backbone; 10μL of 2x NEB Gibson assembly master mix; water to bring entire reaction to 20μL
  4. Gibson reaction was run in a thermocycler at 50°C for 1 hour
  5. 2μL of the above reaction was transformed into NEB 5-alpha competent E.coli
  6. Cells on plates were subsequently screened using standard colony PCR and verification digest methods