Team:UC Chile2/Protocols
From 2012.igem.org
(Difference between revisions)
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</div> | </div> | ||
- | < | + | <h3>Materials</h3> |
<ul> | <ul> | ||
<li>Liquid BG-11 Media</li> | <li>Liquid BG-11 Media</li> | ||
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<li>Antibiotics (according to your selection resistance)</li> | <li>Antibiotics (according to your selection resistance)</li> | ||
<li>DNA for transformation</li> | <li>DNA for transformation</li> | ||
- | + | </ul> | |
- | < | + | <h3>To be prepared previously</h3> |
<br> | <br> | ||
Concentrate the DNA to be transformed into Synechocystis to 1µg/µL in 10µL for each transformation (A total of 10 ug of the transformation plasmid). | Concentrate the DNA to be transformed into Synechocystis to 1µg/µL in 10µL for each transformation (A total of 10 ug of the transformation plasmid). | ||
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Once you have reached the OD730nm and have enough DNA, proceed to transform. | Once you have reached the OD730nm and have enough DNA, proceed to transform. | ||
<br> | <br> | ||
- | < | + | <h3>Transformation</h3> |
All subsequent actions are to be realized under sterile conditions. | All subsequent actions are to be realized under sterile conditions. | ||
- | + | <ul> | |
<li>1.-Transfer Synechocystis cells into 50 mL Falcon tubes and centrifuge 10 minutes at 2760g (Room temperature)</li> | <li>1.-Transfer Synechocystis cells into 50 mL Falcon tubes and centrifuge 10 minutes at 2760g (Room temperature)</li> | ||
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<li>Softly mix contents with the point of the pipet. Repeat the procedure to all but the transformation control tube (without DNA). </li> | <li>Softly mix contents with the point of the pipet. Repeat the procedure to all but the transformation control tube (without DNA). </li> | ||
<li>8.- Leave the tubes in a light chamber (50 uE/s²/m²) at 30°C for 5 hours. Flick the tubes at 2.5 hours. </li> | <li>8.- Leave the tubes in a light chamber (50 uE/s²/m²) at 30°C for 5 hours. Flick the tubes at 2.5 hours. </li> | ||
- | + | </ul> | |
- | < | + | <h3>Plate preparation</h3> |
<br> | <br> | ||
Prepare 50 mL BG-11 plates with no antibiotic for the transformation day. | Prepare 50 mL BG-11 plates with no antibiotic for the transformation day. | ||
Put Millipore membrane filters on BG-11 plates. | Put Millipore membrane filters on BG-11 plates. | ||
- | + | <ul> | |
<li>9.- After the 5 hours of recuperation, take the celular suspension and spread over the Millipore membrane. The suspension is to be spreaded carefully over the whole membrane using a sterile bended Pasteur pipete.</li> | <li>9.- After the 5 hours of recuperation, take the celular suspension and spread over the Millipore membrane. The suspension is to be spreaded carefully over the whole membrane using a sterile bended Pasteur pipete.</li> | ||
<li>10.- Let the membranes dry before transfering the plates to the growth chamber.</li> | <li>10.- Let the membranes dry before transfering the plates to the growth chamber.</li> | ||
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<li>13.- After the 3 days have passed, transfer the membrane to BG-11 plates with full concentration of antibiotic (25ug/mL) [see tabla 2].</li> | <li>13.- After the 3 days have passed, transfer the membrane to BG-11 plates with full concentration of antibiotic (25ug/mL) [see tabla 2].</li> | ||
</ul> | </ul> | ||
- | |||
<br> | <br> | ||
<i>Colonies should appear between 1 to 2 weeks after the transformation. First you will see most of the cellular suspension dissapear and slowly small colonies should appear in the plate.</i> | <i>Colonies should appear between 1 to 2 weeks after the transformation. First you will see most of the cellular suspension dissapear and slowly small colonies should appear in the plate.</i> | ||
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<div id="Reinoculation"> | <div id="Reinoculation"> | ||
<h2>Reinoculation</h2> | <h2>Reinoculation</h2> |
Revision as of 00:29, 24 September 2012