Gibson Assembly Cloning
Suzanne R Pfeffer, Claire Y Chiang
Abstract
Gibson assembly requires a vector backbone and one or more inserts that have been PCR amplified. The inserts should have 15-20 base pairs of overlap at ligation sites, so primers used to amplify the inserts should contain a tail overhang with this homology. The vector backbone should be linear.
A Gibson reaction uses a master mix that can be homemade or commercially purchased. Add the appropriate amount of master mix, then your vector and insert at 2-3 fold molar excess for a 20 µl reaction. Exact ratios will need to be optimized based on the size of your vector and inserts and how many inserts you have. Incubate this reaction at 50°C for 60 minutes. Then transform this reaction into DH5 alpha (or competent cell of choice) and plate on LB agar with appropriate antibiotic.
Steps
Prepare Gibson master mix
Make 5 ml of 5x reaction buffer
25% PEG-8000 1.25 g
500 mM Tris-HCl pH 7.5 2.5 ml
50 mM MgCl2 0.25 ml of 1M
50 mM DTT 0.25 ml of 1M
1 mM each dNTPs 0.05 ml each of 100mM
5 mM NAD 0.5 ml of 50 mM
Prepare master mix
Master Mix
320 µl 5X Reaction Buffer (above)
0.64 µl 10U/µl T5 exonuclease
20 µl 2U/µl Phusion
160 µl 40U/µl Taq Ligase
700 µl Water
Use master mix as 1.5x. Snapfreeze in 15µL aliquots and store at -80°C.
Gibson assembly
Prepare linearized vector backbone by restriction enzyme digestion or PCR amplification.
Prepare inserts by PCR amplification. Ensure that each insert has 15-20 base pairs of overlap with other inserts/vector at ligation sites, added by primer design.
Take a 15 ul aliquot of 1.5x Gibson master mix on ice. Add 50-100 ng of vector with 2-3 fold molar excess of PCR inserts, and sterile water if necessary, to make total reaction volume up to 20µL.
Mix well and let incubate in PCR machine for 1h 0m 0s at 50°C
Plasmid preparation
After incubation is complete, transform into competent cells (DH5α) and plate onto LB agar with appropriate antibiotic. Let this grow at 37°C 1h 0m 0s.
Pick a single colony to grow in 5 ml LB culture at 37°C and purify plasmid to check sequencing.
