Difference between revisions of "Team:Exeter/Project"

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             <li>Plate out 200μl on agar laced with 100μg/ml ampicillin, 50μg/ml kanamycin, 35μg/ml chloramphenicol or 15μg/ml tetracycline as appropriate.</li>
 
             <li>Plate out 200μl on agar laced with 100μg/ml ampicillin, 50μg/ml kanamycin, 35μg/ml chloramphenicol or 15μg/ml tetracycline as appropriate.</li>
 
             <li>Incubate at 37˚C overnight.</li>
 
             <li>Incubate at 37˚C overnight.</li>
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        </ol>
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<h6>KillerRed KillerOrange protocol</h6>
 +
        <ol>
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            <li>Prepare 5ml overnight cultures of KillerRed, KillerOrange and pSB1C3 RFP in BL21 DE3 E.coli. This can be done from a glycerol stock or fresh transformation. Place in 37 C 220rpm incubator overnight.</li>
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            <li>Prepare five 250ml erlenmeyer flasks with 50ml of LB broth and add 50μl of 35mg/ml chloramphenicol to give a concentration of 35μg/ml. Cover the flasks in tin foil.</li>
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            <li>Label the flasks KillerRed induced, KillerRed not induced, KillerOrange induced,                    KillerOrange not induced and pSB1C3 RFP</li>
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            <li>Measure the OD of the overnight cultures and inoculate the 250ml flasks to a starting OD of 0.1 </li>
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            <li>Check the OD periodically until it reaches an OD of 0.4 on a cuvette reader or 0.23 on a tecan spectrophotometer.</li>
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            <li>Once the target OD has been reached, induce the flasks that have been labelled induced with 100μl of 0.1M IPTG. Incubate at 37˚C 220 rpm overnight.</li>
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            <li>Measure the OD and fluorescence of each culture.</li>
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            <li>Prepare thirty 10ml falcon tubes to be used in a serial dilution of 10-3,10-4,10-5. Add 5μl of undiluted culture to 4995μl of LB broth and invert the tube 5-10 times, this is the 10-3 culture. Take 500μl of the 10-3 and add it to 4500μl  of LB broth, invert the tube 5-10 times, this is the 10-4. Take 500μl of the 10-4  and add it to 4500μl of LB broth, invert the tube 5-10 times, this is the 10-5 culture . Repeat this twice for all the samples (KillerRed induced, KillerRed not induced, KillerOrange induced, KillerOrange not induced and pSB1C3 RFP). One set of fifteen samples should be covered in tin foil, the other set left uncovered.</li>
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            <li>Place the falcon tubes in the light box, label down for the uncovered samples.</li>
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            <li>Expose for 6hrs, take the temperature inside the box periodically using a thermocouple.</li>
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            <li>Spread plate 200μl of each sample and incubate at 37˚C overnight.</li>
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            <li>Count the number of colonies on each plate.</li>
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 +
        </ol>
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 +
<h6>Ministat Protocol</h6>
 +
        <ol>
 +
            <li>Prepare the desired number of media containers and culture chambers ready to be autoclaved.(see jove ministat page for details)</li>
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            <li>Inside a flow hood add media to the containers. Add 100μl of inoculum to the sterile culture chamber. Connect the media container to the culture chamber.</li>
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            <li>Arrange the ministat array in the heat block and set the effluent volume to around 35ml by moving the effluent needle up to the desired height in the chamber.</li>
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            <li>Set the peristaltic pump to 90 rpm and allow the chambers to fill. Once at 35 ml turn off the peristaltic pump. Turn on the air pump to start aeration and agitation. Let the culture grow for 24hrs</li>
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            <li>Turn on the peristaltic pump to 7.5 rpm this will give a flow rate of approx 4ml/hr. Ensure that the effluent tubes are uncovered and inside a collecting vessel. </li>
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            <li>Each day take a sample in a sterile container for testing. Glycerol stocks should be taken. These can then be tested later.</li>
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        </ol>
 +
 +
<h6>HGT Protocol</h6>
 +
        <ol>
 +
            <li>Prepare a 5ml overnight of pSB1C3 RFP DH5α in a 10ml falcon tube.</li>
 +
            <li>Prepare working of lysozyme in reaction buffer from enzcheck kit. Two fold dilution of 1000U/ml lysozyme C (Galus Galus) in reaction buffer. 50µl of working solution is needed per reaction.</li>
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            <li>Add 50µl of working solution to 50µl of overnight culture in a PCR tube incubate at room temp for 30 mins</li>
 +
            <li>Incubate overnight at 55 ℃ (15 hours should be enough to denature all the lysozyme)</li>
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            <li>Perform full transformation protocol using 3µl of the lysate, and incubate 3µl in with competent cells for 50 mins before continuing from step six in the transformation protocol.</li>
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            <li>Plate the remaining lysate onto a chloramphenicol plate as a negative control.</li>
 +
            <li>Count colonies, overnight these and measure fluroescence.</li>                   
 
         </ol>
 
         </ol>
  
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</ol>
 
 
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Revision as of 14:11, 27 September 2016