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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>Monday 12<sup>th</sup> August</h3>
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| - | <p><b><em>
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| - | Glycerol Stock, Miniprep, M13 plate test, Patches
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| - | </br></em></b></p>
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| - | <b>Glycerol Stock</b>
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| - | sSP018 - NEB with E1010<br>
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| - | sSP017<br>
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| - | sSP016<br>
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| - | Take 1.5ml 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>Miniprep</b><br>
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| - | E1010<br>
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| - | J04450<br>
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| - | <br>
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| - | 1) Pellet 2x 9ml of liquid culture (4000rpm, 10 min)<br>
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| - | 2) Discard supernatant<br>
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| - | 3) resuspend the cells in 250ul of resuspension olution<br>
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| - | 4) add 250ul of lysis solution, mix by inverting 4-6 times<br>
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| - | 5) add 350ul 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 ul 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 1.5ml 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: ng/ul<br>
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| - | E1010: ng/ul<br>
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| - | <br>
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| - | <br>
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| - | <b>M13 plate test</b><br>
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| - | 1) 100 μ l of plating bacteria per tube<br>
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| - | 2) Prepare tenfold serial dilutions (10-6 to 10-9 ) of the bacteriophage stock in LB<br>
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| - | 3) Put 10 μ l/ 100 μ l of each dilution to the bacteria<br>
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| - | 4) Mix the bacteriophage particles with the bacterial culture by vortexing gently.<br>
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| - | 5) Add 40 μ l of X-gal solution (working conc) and IPTG (working conc) solution to each of the tubes<br>
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| - | 6) Add 3ml of Agar to each tube and gently vortexing for 3 seconds<br>
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| - | 7) Pour the mixture onto plates containing LB medium supplemented with 5 mM MgCl2 <br>
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| - | 8) Swirl the plate gently to ensure an even distribution of bacteria and agar.<br>
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| - | 9) Incubate them at 37°C.<br>
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| - | <br>
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| - | <b>Patches</b><br>
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| - | check them and prepare new ones of those that haven’t been done yet<br>
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| - | </html>
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