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}} | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Gram Staining Protocol|<html> | ||
+ | <ol> | ||
+ | <li> | ||
+ | Transfer 100 ul of sterile distilled water in an eppendorf; | ||
+ | </li> | ||
+ | <li> | ||
+ | Pick up a colony using a tip and resuspend it in the sterile water; | ||
+ | </li> | ||
+ | <li> | ||
+ | Verify that the glass slide is cleaned and degreased or clean it with 70% alcohol; | ||
+ | </li> | ||
+ | <li> | ||
+ | Transfer 20 ul of bacterial suspension on the slide; | ||
+ | </li> | ||
+ | <li> | ||
+ | Swipe gently bacterial suspension with the aid of a sterile loop to occupy 1-2 cm at the center of the slide; | ||
+ | </li> | ||
+ | <li> | ||
+ | Let dry the slide by evaporation; | ||
+ | </li> | ||
+ | <li> | ||
+ | Cover the central part of the slide with methanol, remove the excess and let it evaporate; | ||
+ | </li> | ||
+ | <li> | ||
+ | Sock the slide in the crystal violet solution for 1 min, wash then with sterile water; | ||
+ | </li> | ||
+ | <li> | ||
+ | Sock the slide in the lugol solution for 1 min, wash then with sterile water; | ||
+ | </li> | ||
+ | <li> | ||
+ | Pour Gram bleach solution on the product for 20-30 s, wash then with sterile water; | ||
+ | </li> | ||
+ | <li> | ||
+ | Sock the slide in the safranin solution for 1 min, wash then with sterile water; | ||
+ | </li> | ||
+ | <li> | ||
+ | Finally let dry the slide and observe it with the microscope. | ||
+ | </li> | ||
+ | </ol> | ||
+ | </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 °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> | ||
- | |||
</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 8µl of PCR reaction and 2µl 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 8µl of PCR reaction and 2µl of 6X loading die; 1kb ladder is good). | ||
</html>|pSB1C3-linearization-PCR}} | </html>|pSB1C3-linearization-PCR}} | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Kinetic assay for ethylene production through micro GC|<html> | ||
+ | <p> | ||
+ | An overnight culture was diluited 1:100 and grown until O.D.600 reached 0.5. After that, 3 | ||
+ | ml of culture induced with 5 mM arabinose was placed with a stirrer in a sealed vial (V = | ||
+ | 15 ml) with a pierceable septum. The sample was then connected to the Micro Gas | ||
+ | Chromatograph Agilent 3000A endowed with two colums: a Mol Sieve 5A Plot and a Plot U | ||
+ | column (see the tables for colums and method specifications). | ||
+ | </p> | ||
+ | <table class="tn-sp-table"> | ||
+ | <TR> | ||
+ | <th> | ||
+ | Column | ||
+ | </th> | ||
+ | <th> | ||
+ | Lenght | ||
+ | </th> | ||
+ | <th> | ||
+ | Diameter | ||
+ | </th> | ||
+ | </TR> | ||
+ | <tr> | ||
+ | <td> | ||
+ | Mol Sieve 5A Plot | ||
+ | </td> | ||
+ | <td> | ||
+ | 10 m | ||
+ | </td> | ||
+ | <td> | ||
+ | 0.32 mm | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | Plot U | ||
+ | </td> | ||
+ | <td> | ||
+ | 8 m | ||
+ | </td> | ||
+ | <td> | ||
+ | 0.32 mm | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th colspan = '2'> | ||
+ | Method used | ||
+ | </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | t sample | ||
+ | </th> | ||
+ | <td> | ||
+ | 50 °C | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | t injector | ||
+ | </th> | ||
+ | <td> | ||
+ | 55 °C | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | t column Mol Sieve 5A Plot | ||
+ | </th> | ||
+ | <td> | ||
+ | 110 °C | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | t column Plot U | ||
+ | </th> | ||
+ | <td> | ||
+ | 70 °C | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | p column Mol Sieve 5A Plot | ||
+ | </th> | ||
+ | <td> | ||
+ | 39.16 psi | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | p column Plot U | ||
+ | </th> | ||
+ | <td> | ||
+ | 21.76 psi | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | t injection | ||
+ | </th> | ||
+ | <td> | ||
+ | 40 us | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | t analysis | ||
+ | </th> | ||
+ | <td> | ||
+ | 95 s | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | A measurment was taken every 45 min / 1 h in order to get an overview of the time course | ||
+ | of ethylene production.<br/> | ||
+ | In order to estimate how much gas was taken for each measurement using the settings | ||
+ | described above, a mass flow meter was connected to the micro GC. | ||
+ | During a measurment, a flow of 3 (± 0.15) ml / min was registered. Due to the fact that a | ||
+ | measurment lasts 10 s, the withdrawn volume was 0.5 ml. | ||
+ | </html>|kinetic-ethylene-production}} | ||
}} | }} |
Revision as of 13:03, 1 August 2013