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 --> | ||
+ | |||
+ | <h1>BioBrick cloning</h1> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <p> | ||
+ | Prepare the digestion mix as follow: | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td> | ||
+ | DNA | ||
+ | </td> | ||
+ | <td> | ||
+ | 500 ng | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | 10X NEB Buffer | ||
+ | </td> | ||
+ | <td> | ||
+ | 2.5 ul | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | 10X BSA | ||
+ | </td> | ||
+ | <td> | ||
+ | 2.5 ul | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | E1 | ||
+ | </td> | ||
+ | <td> | ||
+ | 1 ul | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | E2 | ||
+ | </td> | ||
+ | <td> | ||
+ | 1 ul | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | H2O | ||
+ | </td> | ||
+ | <td> | ||
+ | Up to 25 ul | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <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> | ||
+ | <td> | ||
+ | Insert | ||
+ | </td> | ||
+ | <td> | ||
+ | 3 fold excess | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | Vector | ||
+ | </td> | ||
+ | <td> | ||
+ | 40 ng | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | 10X T4 Ligase Buffer | ||
+ | </td> | ||
+ | <td> | ||
+ | 2 ul | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | T4 Ligase | ||
+ | </td> | ||
+ | <td> | ||
+ | 1 ul | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | H2O | ||
+ | </td> | ||
+ | <TD> | ||
+ | Up to 20 ul | ||
+ | </TD> | ||
+ | </tr> | ||
+ | |||
+ | </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> | ||
+ | </html>|subtilis-transformation}} | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Competent cells transformation efficiency kit (registry)|<html> | ||
+ | <a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit" target="_blank">External link (registry)</a> | ||
+ | </html>|Competent-cells-transformation-efficiency}} | ||
+ | |||
+ | <h2>Miscellaneous</h2> | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Parts extraction and transformation (NEB10β).|<html> | ||
+ | <ol> | ||
+ | <li>Label the empty eppendorfs that will contain the parts, including antibiotic resistance, part denomination and position (and on which kit).</li> | ||
+ | <li>Spot the correct well and label it with a pen.</li> | ||
+ | <li>Push a hole with the pin of a micropipette and resuspend the content with 10ul water.</li> | ||
+ | <li>When the color is dark red, wait 1 minute.</li> | ||
+ | <li>Move the re-suspended part into the correct empty eppendorf.</li> | ||
+ | </ol> | ||
+ | </html>|parts-registry-extraction}} | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Wizard® Plus SV Minipreps DNA Purification System Technical Bulletin|<html> | ||
+ | <a href="http://ita.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/0/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Protocol.pdf" target="_blank">Complete protocol</a> (180kb)<br/> | ||
+ | <a href="http://ita.promega.com/~/media/Files/Resources/ProtCards/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Quick%20Protocol.pdf" target="_blank">Quik protocol</a> (72kb) | ||
+ | </html>|miniprep}} | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Biobrick cloning|<html> | ||
<!--biobrick cloning protocol --> | <!--biobrick cloning protocol --> | ||
<p> | <p> | ||
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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. | 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> | </p> | ||
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- | + | </html>|biobrick-cloning}} | |
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Ligation|<html> | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Ligation|<html> |
Revision as of 12:32, 31 July 2013