Team:Macquarie Australia/Protocols/GibsonAssembly


Gibson Assembly Protocol

Before entering the lab, the assigned Gibson Assembly team for the day organised agar plate requirements, concentration calculations and equipment requirements. All gBlocks had arrived and thus Gibson Assembly was performed on all fragments.

Each tube was labelled with antibiotic (C = Chloramphenicol, K = Kanamycin, A = Ampicillin) resistance contained within the allocated plasmid. Tubes labeled 1 & 2 represent Heme Oxygenase fragment mixtures, tubes labeled 3 represent Deinococcus fragment mixtures & tubes labeled 4 & 5 represent Agrobacterium fragment mixtures. Take note that #1C and #4C contain T7 promoter fragments whereas the other BioBricks are T7 'promoter-less'. Deinococcus BioBrick does not contain any T7 promoters as described here.

Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer. The table below shows each tube with vector & volume, volume of Gibson Master Mix, water volume & volume of each gBlock fragment. After incubation, these were transformed into Top10 cells using the Transformation Protocol found here - Transformation Protocol.

#1C #2K #2A #3K #3A #4C #5K #5A
1. Addition of 20ng Gene Block Fragments in appropriate tube 2µl Hemo_T7A 2µl Hemo_A 2µl Hemo_A 2µl Deino_A 2µl Deino_A 2µl Agro_T7A 2µl Agro_A 2µl Agro_A
2µl Hemo_B 2µl Hemo_B 2µl Hemo_B 2µl Deino_B 2µl Deino_B 2µl Agro_B 2µl Agro_B 2µl Agro_B

nil nil nil 2µl Deino_C 2µl Deino_C 2µl Agro_C 2µl Agro_C 2µl Agro_C

nil nil nil 2µl Deino_D 2µl Deino_D 2µl Agro_D 2µl Agro_D 2µl Agro_D

nil nil nil 2µl Deino_E 2µl Deino_E 2µl Agro_E 2µl Agro_E 2µl Agro_E
2. Addition of 0.05 pmol of vector 2.7µl PSB-1C3 2.9µl PSB-1K3 2.8µl PSB-1A3 2.9 µl PSB-1K3 2.8 µl PSB-1A3 2.7µl PSB-1C3 2.9µl PSB-1K3 2.8µl PSB-1A3
3. Addition of Gibson Master Mix (µl) 10 10 10 12.9 12.8 12.7 12.9 12.8
4. Addition of deionised H2O 3.3µl 3.1µl 3.2µl nil nil nil nil nil

5. After addition of all components incubation at 50°C for 60 min followed.

Performing Gibson Assembly
Planning lab work for the day
IMG 6152.JPG