Team:UNITN-Trento/Protocols
From 2013.igem.org
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html> | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html> | ||
- | < | + | <!--biobrick cloning protocol --> |
+ | <p> | ||
+ | Prepare the digestion mix as follow: | ||
+ | </p> | ||
<table> | <table> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
- | + | DNA | |
</td> | </td> | ||
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<td> | <td> | ||
- | + | 500 ng | |
</td> | </td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | + | 10X NEB Buffer | |
</td> | </td> | ||
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<td> | <td> | ||
- | + | 2.5 ul | |
</td> | </td> | ||
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</tr> | </tr> | ||
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<tr> | <tr> | ||
- | + | <td> | |
- | + | 10X BSA | |
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- | <td | + | |
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</td> | </td> | ||
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<td> | <td> | ||
- | + | 2.5 ul | |
</td> | </td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | + | E1 | |
+ | </td> | ||
+ | <td> | ||
+ | 1 ul | ||
</td> | </td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | + | E2 | |
</td> | </td> | ||
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<td> | <td> | ||
- | + | 1 ul | |
</td> | </td> | ||
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</tr> | </tr> | ||
- | + | <tr> | |
<td> | <td> | ||
- | + | H2O | |
+ | </td> | ||
+ | <td> | ||
+ | Up to 25 ul | ||
</td> | </td> | ||
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</tr> | </tr> | ||
+ | </table> | ||
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+ | <p> | ||
+ | Incubate the reaction mix at 37 °C for 30 min. Disactivate then the enzymes incubating the mix at 80 °C for 20 min. | ||
+ | The next step will be the ligation of the digestion products. The raction mix is prepared as follow: | ||
+ | </p> | ||
+ | <table> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | + | Insert | |
+ | </td> | ||
+ | <td> | ||
+ | 3 fold excess | ||
</td> | </td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | + | Vector | |
</td> | </td> | ||
- | < | + | <td> |
- | + | 40 ng | |
- | </ | + | </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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<td> | <td> | ||
- | + | 10X T4 Ligase Buffer | |
</td> | </td> | ||
<td> | <td> | ||
- | + | 2 ul | |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
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<td> | <td> | ||
- | + | T4 Ligase | |
</td> | </td> | ||
<td> | <td> | ||
- | + | 1 ul | |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td | + | <td> |
- | + | H2O | |
</td> | </td> | ||
+ | <TD> | ||
+ | Up to 20 ul | ||
+ | </TD> | ||
</tr> | </tr> | ||
+ | |||
</table> | </table> | ||
- | < | + | <p> |
- | + | Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 °C for 20 min. Transorm 10 ul of the reaction in competent cells. | |
- | + | </p> | |
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- | </ | + | |
</html>|subtilis-transformation}} | </html>|subtilis-transformation}} | ||
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Prepare your reaction and incubate at RT for 2 hours. Transform half of the reaction into 200μL of “homemade” competent cells (DH5α, NEB10β, Novablue or other appropriate strains) following a standard transformation protocol. Plate all the cells. | Prepare your reaction and incubate at RT for 2 hours. Transform half of the reaction into 200μL of “homemade” competent cells (DH5α, NEB10β, Novablue or other appropriate strains) following a standard transformation protocol. Plate all the cells. | ||
</html>|Ligation}} | </html>|Ligation}} | ||
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Digestion|<html> | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Digestion|<html> | ||
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<h4>Classic Cloning - for plasmids</h4> | <h4>Classic Cloning - for plasmids</h4> | ||
Incubate at 37°C overnight. The day after add 1µL of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37°C. | Incubate at 37°C overnight. The day after add 1µL of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37°C. | ||
- | + | <h4>Biobricks Cloning</h4> | |
+ | Incubate at 37°C for 30 minutes. Then disactivate the enzymes at 80°C for 20 minutes. | ||
+ | <h4>Screening</h4> | ||
Incubate for 1.5h at 37°C. Run all the digested product on an agarose gel to screen colonies. | Incubate for 1.5h at 37°C. Run all the digested product on an agarose gel to screen colonies. | ||
<br/><br/><hr/><br/> | <br/><br/><hr/><br/> |
Revision as of 12:23, 31 July 2013