Team:Macquarie Australia/Protocols/GibsonAssembly

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Revision as of 02:22, 11 September 2012

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. This was transformed into Top10 cells using the Transformation Protocol.

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. </center>

	#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

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