Team:Evry/Protocols/02
From 2013.igem.org
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<h2> Goal </h2> | <h2> Goal </h2> | ||
- | <p>In this step, the heat shock (between -80°C up to 42°C) induces bacteria to be in state of competence. DNA from the environment (in | + | <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> |
<h2> Preparation </h2> | <h2> Preparation </h2> | ||
+ | <h3>Chimiocompetent cells</h3> | ||
<p>For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA. If the ligation protocol is a | <p>For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA. If the ligation protocol is a | ||
- | <a href="https://2013.igem.org/Team:Evry/Protocols/05">Golden Gate</a> , add 5 µL instead.<br/> | + | <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/> | ||
Line 21: | Line 22: | ||
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/> | ||
</p> | </p> | ||
+ | <h3>Electrocompetent cells</h3> | ||
<h2> Controles</h2> | <h2> Controles</h2> | ||
<p>To check if the transformation have work, make a positive and a negative controle.<br/> | <p>To check if the transformation have work, make a positive and a negative controle.<br/> |
Latest revision as of 09:56, 2 September 2013
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.
If the transformation have worked, colonies must have a red phenotype.
Without plasmid with antibiotic resistance, no colonies must grow.