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. 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.


Transformation was performed and is described below.

Transformation Protocol:

Before you start: • Prepare an ice bath.
• Prepare a 42 °C water bath.
• Pre-warm SOC buffer and plates at 37 °C.
• Autoclaved 1.5 mL Eppendorf tubes.
1. Incubate both the plasmid preparation and the competent cells on ice for 15 minutes in separate 1.5 mL Eppendorf tubes.
2. Combine 3 μL of plasmid prep. with approximately 500 μL of the competent cells preparation.
3. Heat shock the mixture for 40 seconds at 42 °C, then immediately incubate it on ice for 2 minutes.
4. Following this, add 1.0 mL of pre-warmed SOC buffer to your mixture, transfer to a falcon tube and incubate for 1 hour at 37 °C.
5. Inoculate pre-warmed plates with 100 μL or 300 μL of the cell suspension and incubate overnight at 37 °C.



#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
Calculations
IMG 6152.JPG


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