Team:UC Chile2/Protocols

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<h2>Transformation of Synechocystis PCC. 6803</h2>
<h2>Transformation of Synechocystis PCC. 6803</h2>
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<p><b>Materials</b></p>
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<ul>
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<li>Liquid BG-11 Media</li>
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<li>Solid BG-11 Media</li>
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<li>Sterile 250 mL Erlenmeyer flask</li>
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<li>Sterile 50 mL Falcon tube (2)</li>
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<li>Sterile 1.5ml Eppendorf  tubes</li>
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<li>Petri Dishes</li>
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<li>Millipore Membrane Filters, 0.45um HA (HAWP09000) </li>
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<li>Pasteur pipette</li>
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<li>Antibiotics (according to your selection resistance)</li>
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<li>DNA for transformation</li>
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</ul>
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<p><b>To be prepared previously</b>
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<br>
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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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<br>
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Inoculate Synechocystis PCC 6803 into a sterile 250mL Erlenmeyer flask with 150mL* of liquid BG-11 and grow culture until you reach an OD730nm  of 0.8 – 1.0 (That should take between 6 to 10 days dependending on the amount of initial inoculum).
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* For each 50 mL of Synechocystis PCC 6803 you will have 5 individual transformations.
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<br>
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Once you have reached the OD730nm and have enough DNA, proceed to transform.
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<br>
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<b>Transformation</b>
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All subsequent actions are to be realized under sterile conditions.
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<li>1.-Transfer Synechocystis cells into 50 mL Falcon tubes and centrifuge 10 minutes at 2760g (Room temperature)</li>
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<li>2.- Wash cells: </li>
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Discard supernatant and resuspend pellet in the following manner:
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<ul>- Add 1mL of liquid BG-11 and softly pipet cells until resuspended.</ul>
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<ul>- Add 4mL of liquid BG-11 and pipet cells until completely mixed.</ul>
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<ul>- Add 5ml of BG-11 and pipet cells.</ul>
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* It is important that cells are throroughly resuspended so that recieve fresh media and do not form aggregates.
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<li>3.- Centrifuge 10 minutos at 2760g (Room temperature).</li>
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<li>4.- Discard supernatant and extract any remaining media by pipeting.</li>
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<li>5.- Resuspend pellet with 1mL of BG-11 media. Once resuspended, add 0.5 mL more of BG-11. </li>
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<li>6.- Distribuite 300 uL of resuspended cells into sterile 1,5 mL Eppendorf tube.</li>
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<li>* Make sure to pipet into the bottom of the tube so that none of the cells remain in the walls of the tube.</li>
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<li>7.- Add 10 μL of plasmid DNA (1μg/μL) to the tube containing the 300 uL of the Synechocystis cells.</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>
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<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>
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<b>Plate preparation</b>
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<br>
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Prepare 50 mL BG-11 plates with no antibiotic for the transformation day.
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Put Millipore membrane filters on BG-11 plates.
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<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>
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<li>10.- Let the membranes dry before transfering the plates to the growth chamber.</li>
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<li>11.- Transfer plates to the growth chamber putting the membrane facing up and leave at 30°C for 12 hours in light.</li>
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<li>12.- Transfer membranes to BG-11 plates that contain half of the concentration of the antibiotic (12.5 ug/mL of antibiotic) [see table 2] and leave growing for 3 days at 30°C in light. </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>
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<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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<p>Protocol goes here!</p>
<p>Protocol goes here!</p>
<div id="Reinoculation">
<div id="Reinoculation">

Revision as of 00:25, 24 September 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012