Gibson Assembly

Protocol

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; 10uL of 2x NEB Gibson assembly master mix; water to bring entire reaction to 20uL
4. Gibson reaction was run in a thermocycler at 50C for 1 hour
5. 2uL of the above reaction was transformed into NEB 5-alpha competent E.coli
6. Cells on plates were subsequently screen using standard colony PCR and verification digest methods