Team:Macquarie Australia/Protocols/GibsonAssembly
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Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer </center> | Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer </center> | ||
<br/> | <br/> | ||
+ | <center>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. | ||
+ | </center> | ||
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Revision as of 02:16, 11 September 2012
Gibson Assembly 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.
#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.