Team:Evry/Protocols/02

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<h1 align='center'> Protocol for transformation </h1>
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<p>For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA. IF the ligation protocol is a golden gate, add 5 µL instead.<br/>
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<h1> Transformation </h1>
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<h2> Goal </h2>
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<p>In this step, <h1>à modif</h1>the heat shock (between -80°C up to 42°C) induces chimiocompetent bacteria to be in state of competence. DNA from the environment (in this case: plasmid) can then integrate the cell.</p>
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<h2> Preparation </h2>
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<h3>Chimiocompetent cells</h3>
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<p>For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA. If the ligation protocol is a
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<a href="https://2013.igem.org/Team:Evry/Protocols/05" target='_blank'>Golden Gate</a> , add 5 µL instead.<br/>
Let 30 minutes on ice.<br/>
Let 30 minutes on ice.<br/>
Let the cells at 42°C for exactly 50 seconds.<br/>
Let the cells at 42°C for exactly 50 seconds.<br/>
Let 5 minutes on ice.<br/>
Let 5 minutes on ice.<br/>
Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.<br/>
Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.<br/>
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<h3>Electrocompetent cells</h3>
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<h2> Controles</h2>
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<p>To check if the transformation have work, make a positive and a negative controle.<br/>
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<li>Positive controle: Use plasmid pSB1A3<br/>
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If the transformation have worked, colonies must have a red phenotype.<br/><br/>
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<li>Negative controle: Ø <br/>
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Without plasmid with antibiotic resistance, no colonies must grow. </p>
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Latest revision as of 09:56, 2 September 2013

Iron coli project

Transformation

Goal

In this step,

à modif

the heat shock (between -80°C up to 42°C) induces chimiocompetent bacteria to be in state of competence. DNA from the environment (in this case: plasmid) can then integrate the cell.

Preparation

Chimiocompetent cells

For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA. If the ligation protocol is a Golden Gate , add 5 µL instead.
Let 30 minutes on ice.
Let the cells at 42°C for exactly 50 seconds.
Let 5 minutes on ice.
Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.

Electrocompetent cells

Controles

To check if the transformation have work, make a positive and a negative controle.

  • Positive controle: Use plasmid pSB1A3
    If the transformation have worked, colonies must have a red phenotype.

  • Negative controle: Ø
    Without plasmid with antibiotic resistance, no colonies must grow.

    • Retrieved from "http://2013.igem.org/Team:Evry/Protocols/02"