Team:Macquarie Australia/Protocols/GibsonAssembly
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<center>==='''Gibson Assembly Protocol'''===</center> | <center>==='''Gibson Assembly Protocol'''===</center> | ||
Revision as of 02:22, 11 September 2012
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.