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Transformation Protocol
From 2012.igem.org
(Created page with "Transformation Protocol: <p> Before you start: <p> • Prepare an ice bath. <p> • Prepare a 42 °C water bath. <p> • Pre-warm SOC buffer and plates at 37 °C. <p> • Auto...") |
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Transformation Protocol: | Transformation Protocol: | ||
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Before you start: | Before you start: | ||
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• Prepare an ice bath. | • Prepare an ice bath. | ||
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• Prepare a 42 °C water bath. | • Prepare a 42 °C water bath. | ||
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• Pre-warm SOC buffer and plates at 37 °C. | • Pre-warm SOC buffer and plates at 37 °C. | ||
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• Autoclaved 1.5 mL Eppendorf tubes. | • Autoclaved 1.5 mL Eppendorf tubes. | ||
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• 15 ml falcon tubes. | • 15 ml falcon tubes. | ||
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1. Incubate both the plasmid preparation and the competent cells on ice for 15 minutes in separate 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. | ||
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2. Combine 3 μL of plasmid prep. with approximately 500 μL of the competent cells preparation. | 2. Combine 3 μL of plasmid prep. with approximately 500 μL of the competent cells preparation. | ||
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3. Heat shock the mixture for 40 seconds at 42 °C, then immediately incubate it on ice for 2 minutes. | 3. Heat shock the mixture for 40 seconds at 42 °C, then immediately incubate it on ice for 2 minutes. | ||
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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. | 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. | ||
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5. Inoculate pre-warmed plates with 100 μL or 300 μL of the cell suspension and incubate overnight at 37 °C. | 5. Inoculate pre-warmed plates with 100 μL or 300 μL of the cell suspension and incubate overnight at 37 °C. | ||
Revision as of 05:30, 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.
• 15 ml falcon 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.
