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| - | <div class ="tbnote">
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| - | <h2>Phage Sensor</h2>
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| - | <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Phage_Sensor" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
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| - | <div style="clear: both;"></div>
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| - | <h3>Friday 9<sup>th</sup> August</h3>
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| - | <p><b><em>
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| - | Liquid culture, Gel,
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| - | Trafo of BBa_E1010 (pSB1C3 backbone) into NEB turbo, Glycerol Stock, Making electro competent cells of pir+ E.coli, Transforming pKD13 into pir+ E.coli, Miniprep NEB turbo with BBa-J04450:, Liquid culture and Patch plates.
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| - | </br></em></b></p>
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| - | <br><b>Liquid culture</b><br>
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| - | 1:1000 from liquid: FR-433 (M13), 10 ml<br>
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| - | pir+ E.coli: from plate, 25 ml<br>
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| - | XL10: from plate 10 ml<br>
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| - | <br>
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| - | <b>Gel</b>: rest of circular backbone PCR product, 1% Agar, 100V, 20 min<br>
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| - | SPCR4-7-8-9-10 (40.5) SPCR4-7-8-9-10 (60.5)<br>
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| - | Gel picture: <br>
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| - | didn’t work<br>
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| - | <br>
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| - | <b>Trafo of BBa_E1010 (pSB1C3 backbone) into NEB turbo</b><br>
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| - | 1) Thaw competent cells on ice.<br>
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| - | 2) Place 20 µl of cells in a pre-chilled Eppendorf tube.<br>
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| - | 3) Add 2.5 µl of plasmid (from Biobrick stock)<br>
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| - | 4) Mix gently by flicking the tube.<br>
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| - | 5) Chill on ice for 10 minutes.<br>
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| - | 4) Heat shock at 42 °C for 30 seconds <br>
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| - | 5) Return to ice for 2 minutes.<br>
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| - | 6) Add 200 µl LB medium and recover the cells by shaking at 37 °C.<br>
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| - | Chloramphenicol: 60-120 minutes<br>
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| - | 7) Plate out the cells on selective LB (10ul)<br>
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| - | 8) Incubate at 37 °C. Transformants should appear within 12 hrs.<br>
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| - | <br>
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| - | <br>
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| - | <b>Glycerol Stock FR-433 (M13) -> sSP012</b><br>
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| - | Glycerol Stock pir+ -> sSP013<br>
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| - | Glycerol Stock pKD13 -> sSP014<br>
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| - | Glycerol Stock NEB turbo with BBa-J04450 -> sSP015<br>
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| - | Take 1.5 ml of ON culture + 500ul Glycerol (60%)<br>
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| - | Freeze at -80°C<br>
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| - | <br>
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| - | <b>Making electro competent cells of pir+ E.coli</b><br>
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| - | Preparation of Electrocompetent Cells<br>
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| - | Note: Competent cells should never be vortexed, as this will cause them to lyse and release salts into the media. Resuspend cells by pipetting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.<br>
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| - | <br>
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| - | 1) The night before the transformation, start an overnight culture of cells.<br>
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| - | pir+ E.coli<br>
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| - | 2) The day of the transformation, dilute the cells 100X.<br>
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| - | 100 ml LB<br>
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| - | Grow at 30°C for about 90 minutes. <br>
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| - | 3) When the cells reach an OD600 of 0.2. <br>
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| - | 4) Harvest the cells.<br>
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| - | When the cells reach an OD600 of between 0.6 and 0.8.<br>
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| - | Split the culture into 2x 50 ml falcon tubes, on ice.<br>
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| - | Centrifuge at 4 °C for 10 min at 4000 rpm.<br>
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| - | 5) Wash and combine the cells.<br>
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| - | Remove the supernatant.<br>
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| - | Resuspend the cells in 2x 25 ml of ice cold water.<br>
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| - | Combine the volumes in a single 50 ml falcon tube.<br>
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| - | 6) Wash the cells 2 more times.<br>
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| - | Centrifuge at 4 °C for 10 min at 4000 rpm.<br>
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| - | Resuspend in 50 ml of ice cold water.<br>
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| - | Repeat.<br>
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| - | 7) Wash and concentrate the cells for electroporation.<br>
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| - | Centrifuge at 4 °C for 10 min at 4000 rpm.<br>
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| - | Resuspend in 1-2 ml of ice cold water.<br>
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| - | We will use 200 µl of washed cells per transformation.<br>
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| - | <br>
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| - | <b>Transforming pKD13 into pir+ E.coli</b><br>
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| - | Add Cells and plasmid into a Electro transformation tube<br>
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| - | Use E.coli 2 for the electro shock<br>
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| - | Plate 200 µl<br>
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| - | <br>
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| - | <b>Miniprep NEB turbo with BBa-J04450:</b><br>
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| - | 1) Pellet 2x 9 ml of liquid culture (4000rpm, 10 min)<br>
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| - | 2) Discard supernatant<br>
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| - | 3) resuspend the cells in 250 µl of resuspension solution<br>
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| - | 4) add 250 µl of lysis solution, mix by inverting 4-6 times<br>
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| - | 5) add 350 µl of neutralization solution<br>
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| - | 4) centrifuge for 5 min<br>
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| - | 5) transfer supernatant to spin column<br>
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| - | 6) centrifuge for 1 min<br>
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| - | 7) discard flow through<br>
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| - | 8) add 500 µl wash solution and centrifuge for 1 min , discard flow through(repeat this step)<br>
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| - | 9) centrifuge for 1 min to remove left over liquid<br>
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| - | 10) transfer the column on a 1.5 ml tube<br>
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| - | 11) add 50ul of elution buffer and incubate for 2 min<br>
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| - | 12) centrifuge for 2 min<br>
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| - | 13) Nanodrop the concentration and freeze at -20°<br>
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| - | <br>
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| - | J04450: 0ng/ul<br>
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| - | <br>
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| - | <b>Liquid culture of:</b>
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| - | sSP012<br>
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| - | sSP008<br>
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| - | sSP009<br>
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| - | sSP010<br>
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| - | sSP011<br>
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| - | sSP001<br>
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| - | sSP002<br>
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| - | XL-10 = sSP016<br>
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| - | sSP017= Litmus<br>
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| - | <br>
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| - | <b>Patch plates of:</b><br>
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| - | sSP010<br>
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| - | sSP013<br>
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| - | sSP009s<br>
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| - | sSP014<br>
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| - | sSP012<br>
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