Team:UNITN-Trento/Protocols

From 2013.igem.org

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<a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit" target="_blank">External link (registry)</a>
<a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit" target="_blank">External link (registry)</a>
</html>|Competent-cells-transformation-efficiency}}
</html>|Competent-cells-transformation-efficiency}}
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Gram Staining Protocol|<html>
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<ol>
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<li>
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Transfer 100 ul of sterile distilled water in an eppendorf;
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</li>
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<li>
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Pick up a colony using a tip and resuspend it in the sterile water;
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</li>
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<li>
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Verify that the glass slide is cleaned and degreased or clean it with 70% alcohol;
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</li>
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<li>
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Transfer 20 ul of bacterial suspension on the slide;
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</li>
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<li>
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Swipe gently bacterial suspension with the aid of a sterile loop to occupy 1-2 cm at the center of the slide;
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</li>
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<li>
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Let dry the slide by evaporation;
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</li>
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<li>
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Cover the central part of the slide with methanol, remove the excess and let it evaporate;
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</li>
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<li>
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Sock the slide in the crystal violet solution for 1 min, wash then with sterile water;
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</li>
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<li>
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Sock the slide in the lugol solution for 1 min, wash then with sterile water;
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</li>
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<li>
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Pour Gram bleach solution on the product for 20-30 s, wash then with sterile water;
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</li>
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<li>
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Sock the slide in the safranin solution for 1 min, wash then with sterile water;
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</li>
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<li>
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Finally let dry the slide and observe it with the microscope.
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</li>
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</ol>
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</html>|gram-staining}}
<h2>Miscellaneous</h2>
<h2>Miscellaneous</h2>
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Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 &deg;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 &deg;C for 20 min. Transorm 10 ul of the reaction in competent cells.
</p>
</p>
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</html>|biobrick-cloning}}
</html>|biobrick-cloning}}
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Then run the PCR samples on a 1% agarose gel to verify the success of the reactions (each sample is prepared with 8&micro;l of PCR reaction and 2&micro;l of 6X loading die; 1kb ladder is good).
Then run the PCR samples on a 1% agarose gel to verify the success of the reactions (each sample is prepared with 8&micro;l of PCR reaction and 2&micro;l of 6X loading die; 1kb ladder is good).
</html>|pSB1C3-linearization-PCR}}
</html>|pSB1C3-linearization-PCR}}
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Kinetic assay for ethylene production through micro GC|<html>
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<p>
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An overnight culture was diluited 1:100 and grown until O.D.600 reached 0.5. After that, 3
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ml of culture induced with 5 mM arabinose was placed with a stirrer in a sealed vial (V =
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15 ml) with a pierceable septum. The sample was then connected to the Micro Gas
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Chromatograph Agilent 3000A endowed with two colums: a Mol Sieve 5A Plot and a Plot U
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column (see the tables for colums and method specifications).
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</p>
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<table class="tn-sp-table">
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<TR>
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<th>
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Column
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</th>
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<th>
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Lenght
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</th>
 +
<th>
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Diameter
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</th>
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</TR>
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<tr>
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<td>
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Mol Sieve 5A Plot
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</td>
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<td>
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10 m
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</td>
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<td>
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0.32 mm
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</td>
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</tr>
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<tr>
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<td>
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Plot U
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</td>
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<td>
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8 m
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</td>
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<td>
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0.32 mm
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</td>
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</tr>
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</table>
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<table class="tn-sp-table">
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<tr>
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<th colspan = '2'>
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Method used
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</th>
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</tr>
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<tr>
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<th>
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t sample
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</th>
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<td>
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50 &deg;C
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</td>
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</tr>
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<tr>
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<th>
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t injector
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</th>
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<td>
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55 &deg;C
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</td>
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</tr>
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<tr>
 +
<th>
 +
t column Mol Sieve 5A Plot
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</th>
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<td>
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110 &deg;C
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</td>
 +
</tr>
 +
<tr>
 +
<th>
 +
t column Plot U
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</th>
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<td>
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70 &deg;C
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</td>
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</tr>
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<tr>
 +
<th>
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p column Mol Sieve 5A Plot
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</th>
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<td>
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39.16 psi
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</td>
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</tr>
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<tr>
 +
<th>
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p column Plot U
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</th>
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<td>
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21.76 psi
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</td>
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</tr>
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<tr>
 +
<th>
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t injection
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</th>
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<td>
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40 us
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</td>
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</tr>
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<tr>
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<th>
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t analysis
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</th>
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<td>
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95 s
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</td>
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</tr>
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</table>
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A measurment was taken every 45 min / 1 h in order to get an overview of the time course
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of ethylene production.<br/>
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In order to estimate how much gas was taken for each measurement using the settings
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described above, a mass flow meter was connected to the micro GC.
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During a measurment, a flow of 3 (± 0.15) ml / min was registered. Due to the fact that a
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measurment lasts 10 s, the withdrawn volume was 0.5 ml.
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</html>|kinetic-ethylene-production}}
}}
}}

Revision as of 13:03, 1 August 2013


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