Team:Macquarie Australia/Protocols/GibsonAssembly

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Gibson Assembly Protocol

Before entering the lab, the assigned Gibson Assembly team for the day organised agar plate requirements, concentration calculations and equipment requirements.
Each tube was labelled with antibiotic (C = chloramphenicol, K = kanamycin, A = ampicillin).
#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