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| − | | + | <!-- CONTENT GOES HERE :D --> |
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| − | <h1 style="font-family:Garamond"> Protocols </h1>
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| − | </style>
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| − | <div id="wrap">
| + | |
| − | <ul class="navbar">
| + | |
| − | <li>
| + | |
| − | <a href="https://2015.igem.org/Team:York/Protocols/Growth_Media">Growth Media</a>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="https://2015.igem.org/wiki/index.php?title=Team:York/Protocols/CompetentCells">Competent Cells</a>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="#">Transformations</a>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <a href="#" >Transformation Efficiency Tests</a>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="#">Plasmids</a>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="https://2015.igem.org/Team:York/Protocols/PCR">PCR</a>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <a href="#">General</a>
| + | |
| − | </li>
| + | |
| − |
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="#">Gel Extractions</a>
| + | |
| − |
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <a href="https://2015.igem.org/Team:York/Protocols/PhosphateAssay">Phosphate Assay</a>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <a href="https://2015.igem.org/Team:York/Protocols/PhosphateAssay_Glassmilk">Glassmilk</a>
| + | |
| − |
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <h2>Lysogeny Broth</h2>
| + | |
| − | | + | |
| − | <h3>Materials</h3>
| + | |
| − | <ul>
| + | |
| − | <li>10g of tryptone</li>
| + | |
| − | <li>5g of yeast extract</li>
| + | |
| − | <li>10g of NaCl</li>
| + | |
| − | <li>1L of Deionized Water</li>
| + | |
| − | <li> 1M NaCl </li>
| + | |
| − | <li> 1M KOH </li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <h3>Procedure</h3>
| + | |
| − | <ol>
| + | |
| − | <li>Use a container with at least double the volume of the liquid that you are making.</li>
| + | |
| − | <li>Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water. </li>
| + | |
| − | <li>Adjust the pH of the medium to 7.0 using 1M NaOH or KOH and bring volume up to 1 liter. </li>
| + | |
| − | <li>Autoclave. </li>
| + | |
| − | <li>Store at room temperature or +4°C.</li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | <h2>LB Agar</h2>
| + | |
| − | <h4>Procedure</h4>
| + | |
| − | <ol>
| + | |
| − | <li>follow steps to make lysogeny broth as above</li>
| + | |
| − | <li>add 15g of agar powder per litre of LB</li>
| + | |
| − | <li>autoclave in 200 or 250 mL aliquots in 500mL duran bottles</li>
| + | |
| − | <li>add antibiotic desired to melted agar (~55°C)</li>
| + | |
| − | <li>pour into petri dishes and leave to harden in asceptic fume hood</li>
| + | |
| − | <li>invert, label, wrap with parafilm and store at 4°C</li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | <h2>X-Gal/IPTG Protocol</h2>
| + | |
| − | <p>add the following to 200 mL of melted (~55°C) LB-Agar</p>
| + | |
| − | <ul><li>20 uL X-Gal (20mg/ml)</li>
| + | |
| − | <li>20 uL IPTG (100mM)</li>
| + | |
| − | <li>20 uL of appropriate antibiotic</li>
| + | |
| − | </ul>
| + | |
| − | <p>swirl to mix, try to avoid making bubbles</p>
| + | |
| − | <p>dry at room temp and wrap in foil to prevent degradation</p>
| + | |
| − | | + | |
| − | | + | |
| − | <h2>10X TAE (Tris Acetate EDTA) Buffer</h2>
| + | |
| − | <ul>Dissolve the following in 600mL of dH<sub>2</sub>O</ul>
| + | |
| − | <ol><li> 48.4 g Tris base (FW 121)</li>
| + | |
| − | <li>11.42 mL glacial acetic acid</li>
| + | |
| − | <li>40 mL of 0.25M EDTA (pH 8.0) </li>
| + | |
| − | </ol></ul>
| + | |
| − | <ul>Make up volume to 1.0 L with dH<sub>2</sub>O</ul>
| + | |
| − | <ul>Autoclave in appropriately sized bottle </ul>
| + | |
| − | | + | |
| − | <h2>Agarose Gel Electrophoresis</h3>
| + | |
| − | <ol>
| + | |
| − | <li>add 1g of agarose per 100 mL of TAE (1X) buffer</li>
| + | |
| − | <li>microwave to dissolve</li>
| + | |
| − | <li>add 10 uL of Syber-Safe per 100 mL of TAE buffer</li>
| + | |
| − | <li>pour into gel tray and allow to set (don't forget the comb!) </li>
| + | |
| − | <li>put gel into the tank and fill tank to cover top of gel </li>
| + | |
| − | <li>load ladder and samples and run! </li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | <h2>Competent Cells</h2>
| + | |
| − | <h3>Materials</h3>
| + | |
| − | <ul>
| + | |
| − | <li>LB media (in 50 mL in 250 conical flask) pre- autoclaved (x2)</li>
| + | |
| − | <li>overnight culture of DH5α (x2)</li>
| + | |
| − | <li>shaker in 37°C room</li>
| + | |
| − | <li>15 mL falcon tubes (~x15)</li>
| + | |
| − | <li>eppendorf tubes (1 mL)</li>
| + | |
| − | <li>50% glycerol</li>
| + | |
| − | <li>0.1 M CaCl2 </li>
| + | |
| − | <li>waste bucket with Vircon (kill bacteria)</li>
| + | |
| − | <li>ice buckets</li>
| + | |
| − | </ul>
| + | |
| − | <h3>Protocol:</h3>
| + | |
| − | <ol>
| + | |
| − | <li>inoculate 1% inoculum from overnight culture</li>
| + | |
| − | <li>1 mL for 100 mL medium</li>
| + | |
| − | <li>2 separate 250 mL flasks each with 50 mL LB</li>
| + | |
| − | <li>0.5 mL of culture- grow with shaking until ~ 0.375 OD (at 600 nm) - we went a bit over, but other protocols say this is ok</li>
| + | |
| − | <li>put flasks on ice for 10 minutes</li>
| + | |
| − | <li>put 10 mL of inoculum into 15 mL falcon tubes (repeat to use up all the inoculum)</li>
| + | |
| − | <li>centrifuge @ 5K for 10 minutes @ 4°C</li>
| + | |
| − | <li>discard supernatant and resuspend pellet in 2 mL of 0.1 M CaCl2</li>
| + | |
| − | <li>chill on ice for 20 mins </li>
| + | |
| − | <li>centrifuge @ 5K for 10 minutes @ 4°C</li>
| + | |
| − | <li>discard supernatant and resuspend each pellet in 0.28 mL of 0.1 M CaCl2 and 0.12 mL of 50% glycerol - you can also prepare a solution of CaCl2 and glycerol before hand</li>
| + | |
| − | <li>aliquot 100 μL of resuspension into sterile eppendorfs</li>
| + | |
| − | <li>store at -80°C</li>
| + | |
| − | | + | |
| − | <h2> Colony PCR Protocols</h2>
| + | |
| − | <h4>For PCR reactions up to 50 μL add the following to each PCR Tube: </h4>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>Substance</td>
| + | |
| − | <td>GoTaq</td>
| + | |
| − | <td>Q5</td>
| + | |
| − | <td>Phusion</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Forward Primer</td>
| + | |
| − | <td>2.5</td>
| + | |
| − | <td>2.5</td>
| + | |
| − | <td>2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reverse Primer</td>
| + | |
| − | <td>2.5</td>
| + | |
| − | <td>2.5</td>
| + | |
| − | <td>2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Polmerase</td>
| + | |
| − | <td>.2</td>
| + | |
| − | <td>25 μL mastermix</td>
| + | |
| − | <td>.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>dNTPs</td>
| + | |
| − | <td>.5</td>
| + | |
| − | <td>-</td>
| + | |
| − | <td>1</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>dH<sub>2</sub>O</td>
| + | |
| − | <td>14.3</td>
| + | |
| − | <td>18.0</td>
| + | |
| − | <td>33.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Buffer</td>
| + | |
| − | <td>5</td>
| + | |
| − | <td>-</td>
| + | |
| − | <td>10</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>DNA</td>
| + | |
| − | <td>touch of colony</td>
| + | |
| − | <td>touch of colony</td>
| + | |
| − | <td>touch of colony</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | | + | |
| − | | + | |
| − | <h3>PCR Machine Cycling Times and Temperatures: <i>E. coli</i> and <i>Sinorhizobium Plasmids</i></h3>
| + | |
| − | | + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td colspan="2">Step</td>
| + | |
| − | <td>Temperature</td>
| + | |
| − | <td>Time</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td colspan="2">Initial Denaturation</td>
| + | |
| − | <td>95</td>
| + | |
| − | <td>5:00 min</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td rowspan="3"><br><br>30 Cycles</td>
| + | |
| − | <td>Denaturation</td>
| + | |
| − | <td>95</td>
| + | |
| − | <td>15 sec</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Annealing</td>
| + | |
| − | <td>58</td>
| + | |
| − | <td>15 sec</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Extension</td>
| + | |
| − | <td>72</td>
| + | |
| − | <td>2-4 (1 min per kb)</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td colspan="2">Final Extension</td>
| + | |
| − | <td>72</td>
| + | |
| − | <td>5-10 min</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | | + | |
| − | Load on 1% agarose gels and run electrophoresis tank until the coloured bands are near the bottom of the gel. Use U:genius machine to photograph and visualise bands.
| + | |
| | </p> | | </p> |
| − | | + | <br><br> |
| − | <h2>Digestion Protocols</h2>
| + | |
| − | | + | |
| − | For each of the following digestions add:
| + | |
| − | <ol>
| + | |
| − | <li>1 uL Restriction Enzyme</li>
| + | |
| − | <li>1 ug DNA</li>
| + | |
| − | <li>5 uL 10X NEBuffer</li>
| + | |
| − | <li>dH<sub>2</sub>O to make up 50 uL volume</li>
| + | |
| − | </ol>
| + | |
| − | <p>For the digestions with SpeI, XbaI, PstI and EcoRI-HF, incubation time is 5-15 minutes at 37°C. Inactivation of XbaI and EcoRI-HF takes 20 minuties at 65°C. SpeI and PstI are inactivated at 80°C for 20 minutes. SpeI, XbaI ,and EcoRI-HF have 100% Buffer activity with either NEBuffer 2.1 or Cutsmart while PstI digestions work best with 3.1</p>
| + | |
| − | | + | |
| − | <h2>Mini-Prep or Plasmid Purification</h2>
| + | |
| − | <ul>
| + | |
| − | <li>Harvest bacterial cells<br>
| + | |
| − | 1. Pellet 20ml of saturated E. coli for 60 seconds at 11,000 x g.<br>
| + | |
| − | 2. Discard supernatant and remove as much liquid as possible.</li>
| + | |
| − | <li>Lyse cells<br>
| + | |
| − | 1. Add 500ml resuspension buffer P1 and resuspend cell pellet by vortexing.<br>
| + | |
| − | 2. Split the solution into two 1.5ml microcentrifuge tubes.<br>
| + | |
| − | 3. Add 250μl lysis buffer 2. <br>
| + | |
| − | 4. Mix gently by inverting tube 8 times. <br>
| + | |
| − | 5. Incubate at room temperature for five minutes or until lysate appears clear.<br>
| + | |
| − | 6. Add 300μl neutralization Buffer 3.<br>
| + | |
| − | 7. Mix thoroughly by inverting tube 8 times.</li>
| + | |
| − | <li>Clarification of lysate<br>
| + | |
| − | 1. Centrifuge for 5 minutes at 11,000 x g at room temperature<br>
| + | |
| − | 2. Put 500μl of Buffer PW1 per 1.5ml microcentrifuge tube used in heat block heated to 50<sup>օ</sup>C</li>
| + | |
| − | <li>Bind DNA<br>
| + | |
| − | 1. Place ISOLATE II Plasmid Mini Spin Column in a 2ml Collection Tube<br>
| + | |
| − | 2. Pipette a maximum of 750μl of clarified sample supernatant onto column<br>
| + | |
| − | 3. Incubate at room temperature for 2 minutes.<br>
| + | |
| − | 4. Centrifuge for 1 minute at 11,000 x g and discard flow-through.<br>
| + | |
| − | 5. Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarified sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.</li>
| + | |
| − | <li>Wash silica membrane<br>
| + | |
| − | 1. Add 500μl Wash Buffer Pw1<br>
| + | |
| − | 2. Centrifuge for 1 minute at 11,000 x g <br>
| + | |
| − | 3. Add 600μl Wash Buffer PW2 (supplemented with ethanol)<br>
| + | |
| − | 4. Centrifuge for 1 minute at 11,000 x g <br>
| + | |
| − | 5. Discard flow-through and reuse Collection Tube</li>
| + | |
| − | <li>Dry silica membrane<br>
| + | |
| − | 1. Centrifuge for 2 minutes at 11,000 x g, to remove residual ethanol<br>
| + | |
| − | 2. Place ISOLATE II Plasmid Mini Spin Column in a 1.5ml microcentrifuge tube.</li>
| + | |
| − | <li>Elute DNA<br>
| + | |
| − | 1. Add 50μl Elution Buffer P directly on the top of the silicon matrix<br>
| + | |
| − | 2. Incubate at room temperature for 2 minutes<br>
| + | |
| − | 3. Centrifuge for one minute at 11,000 x g.</li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | | + | |
| − | <div id="phosphate assay">
| + | |
| − | <h2 id="phosphate assay"> Phosphate Assay</h2>
| + | |
| − | <h3> Reagent preparation</h3>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <spam style="font-color:orange">Phosphate Reagent:</spam> 15 ml of colorimetric dye. Ready to use as supplied. Equilibrate to room temperature before use. There may be a small amount of precipitate visible which does not affect the assay performance. Store at room temperature. Actually orange.
| + | |
| − | | + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <span style="font-color:orange">Phosphate Standard: </span>500uL of 10mM Phosphate standard. Ready to use as supplied. Equilibrate to room temperature before use. Store at room temperature. Actually Clear.
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <h3> Standard preparation</h3>
| + | |
| − | <h4>Materials</h4>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <span style="color:green">Phosphate Standard
| + | |
| − |
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <span style="color:blue">Phosphate Reagent
| + | |
| − | | + | |
| − | </li>
| + | |
| − | <li>Distilled Water</li>
| + | |
| − | </ul>
| + | |
| − | </h4>
| + | |
| − | <h4>Procedure</h4>
| + | |
| − | <ol>
| + | |
| − | <li> Dilute 5ul of supplied Phosphate Standard into 495ul of dH2O in a 1.5ml Eppie. Label ‘S’ </li>
| + | |
| − | <li> Make further dilutions to construct the curve. Suggested values are below. Make these in 1.5ml Eppendorfs</li>
| + | |
| − | </ol>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>Volume S/ ul</td>
| + | |
| − | <td>Volume dH2O/ ul</td>
| + | |
| − | <td>Concentration/ nMol/200ul(well)</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>0</td>
| + | |
| − | <td>600</td>
| + | |
| − | <td>0</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>30</td>
| + | |
| − | <td>570</td>
| + | |
| − | <td>1</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>60</td>
| + | |
| − | <td>540</td>
| + | |
| − | <td>2</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>90</td>
| + | |
| − | <td>510</td>
| + | |
| − | <td>3</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>120</td>
| + | |
| − | <td>480</td>
| + | |
| − | <td>4</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>150</td>
| + | |
| − | <td>450</td>
| + | |
| − | <td>5</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <ol>
| + | |
| − | <li>Pipette 200ul of each curve sample into a well.</li>
| + | |
| − | <li>Add 30ul of Phosphate Reagent to each well</li>
| + | |
| − | <li>Leave to equilibrate for 30 mins at room temperature. N.B the reagent is light sensitive so store in a dark room while equilbrating</li>
| + | |
| − | </ol>
| + | |
| − | <h3>Sample preparation</h3>
| + | |
| − | <p>N.B. Make sure our sample is diluted to ensure the readings are within the standard value range.</p>
| + | |
| − | <p>
| + | |
| − | <u>Steps for cell (adherent or suspension) samples:</u>
| + | |
| − | </p>
| + | |
| − | <ol>
| + | |
| − | <li>Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10^6 cells).</li>
| + | |
| − | <li>Wash cells with cold TBS (kept in 4oC fridge, stable for 3 months). </li>
| + | |
| − | <li>Resuspend the cell pellet in 150 µL of Assay Buffer (TBS) on ice. </li>
| + | |
| − | <li>Homogenize cells quickly by pipetting up and down a few times. </li>
| + | |
| − | <li>Sonicate cells for 50 seconds 3X at high setup (one cycle = 30 s sonication – 10 s break – 10 s sonication – 10 s break). </li>
| + | |
| − | <li>Centrifuge sample for 15 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material. </li>
| + | |
| − | <li>Collect supernatant and transfer to a clean tube. </li>
| + | |
| − | </ol>
| + | |
| − | <ul>
| + | |
| − | <li>Keep on ice</li>
| + | |
| − | </ul>
| + | |
| − | <p>
| + | |
| − | <u>Tris-Buffered Saline (TBS) Recipe</u>
| + | |
| − | </p>
| + | |
| − | <ol>
| + | |
| − | <li>50 mM Tris-Cl, pH 7.5</li>
| + | |
| − | <li>150 mM NaCl</li>
| + | |
| − | <li>To prepare, dissolve 6.05 g and 8.76 g NaCl in 800 mL of H2O. </li>
| + | |
| − | <li>Adjust pH to 7.5 with 1 M HCl </li>
| + | |
| − | <li>Tris Make volume up to 1 L with H2O.</li>
| + | |
| − | <p>N.B. TBS is stable at 4°C for 3 mo.</p>
| + | |
| − | </ol>
| + | |
| − | <h3>Assay procedure and detection</h3>
| + | |
| − | <p> Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate.</p>
| + | |
| − | <p>Set up Reaction wells: </p>
| + | |
| − | <ol>
| + | |
| − | <li>Standard wells = 200 µL standard dilution. </li>
| + | |
| − | <li>Sample wells = 1 – 100 µL samples (adjust volume to 200 µL/well with ddH2O). </li>
| + | |
| − | <li>Background control sample wells = 1 – 100 samples (adjust volume to 200 µL/well with ddH2O). </li>
| + | |
| − | <li>Add 30 µL of Phosphate Reagent to standard, sample and background control wells. </li>
| + | |
| − | <li>Mix and incubate at room temperature for 30 minutes protected from light. </li>
| + | |
| − | <li>Measure output at OD = 650 nm on a microplate reader.</li>
| + | |
| − | </ol>
| + | |
| − |
| + | |
| − |
| + | |
| − | <h3> Glassmilk recipe </h3>
| + | |
| − | <ol> <li>Stir 400g silica 325 in 800 ml of dH2O</li>
| + | |
| − | <li>Leave to settle for 90 minutes</li>
| + | |
| − | <li>Remove supernatant and centrifuge 6000 rpm for 10 minutes</li>
| + | |
| − | <li>Resuspend pellet in 250 ml dH2O</li>
| + | |
| − | <li>Add conc. Nitric Acid to 50%</li>
| + | |
| − | <li>Stir and heat to a boil gently</li>
| + | |
| − | <li>Stir to RT gently</li>
| + | |
| − | <li>Centrifuge @ 6000 rpm for 10 minutes (make sure you use either glass or polypropylene tubes, polystyrene tubes can’t withstand over 3000g) </li>
| + | |
| − | <li>Resuspend in 250 ml dH2O</li>
| + | |
| − | <li>Repeat spinning and washing until pH is neutral</li>
| + | |
| − | <li>Resuspend pellet to make 50% by volume slurry</li>
| + | |
| − | </ol>
| + | |
| − | <p>Aliquot and store indefinitely at RT</p>
| + | |
| − |
| + | |
| − | | + | |
| − | <h2>Growth Assay - Plate Reader</h2>
| + | |
| − | | + | |
| − | <h4>Materials</h4>
| + | |
| − | <ul>
| + | |
| − | <li>96 well plate</li>
| + | |
| − | <li>10x MOPS Media</li>
| + | |
| − | <ul><li>dilute to 1X</li></ul>
| + | |
| − | <li>K<sub>2</sub>HPO<sub>4</sub> (dipotassium phosphate)</li>
| + | |
| − | <ul><li>@0.1 mM for low P</li>
| + | |
| − | <li>@1.32 mM for high P - suggested by MOPS media recipe</li>
| + | |
| − | <li>Molar mass- 174.2 g/mol </li>
| + | |
| − | </ul>
| + | |
| − | <li>4 mg/mL glucose</li>
| + | |
| − | <li>strains of bacteria</li>
| + | |
| − | </ul>
| + | |
| − | <h4>Purpose</h4>
| + | |
| − | <p>-used to measure bacterial density by tracking absorbance at intervals of 30 minutes for 48 hours (220 rpm, 37°C) </p>
| + | |
| − | | + | |
| − | <h4>Procedure</h4>
| + | |
| − | <p>Day 1</p>
| + | |
| − | <ol>
| + | |
| − | <li>prepare the phosophate concentrations and filter sterilize them</li>
| + | |
| − | <li>prepare the glucose solution and filter sterilize them</li>
| + | |
| − | <li>prepare 1X MOPS (from 10X solution) filter sterilize them </li>
| + | |
| − | <li>prepare overnights</li>
| + | |
| − | <ol><li>5ml of LB in a BD falcon tube</li>
| + | |
| − | <li>take a colony off the plate with a sterile toothpick and put in falcon tube </li>
| + | |
| − | <li>leave overnight at 37°C in a shaker </li></ol></ol>
| + | |
| − | <p>Day 2</p>
| + | |
| − | <ol>
| + | |
| − | <li>transfer overnight culture into 1.5 mL eppendorf tubes</li>
| + | |
| − | <li>spin down overnights - 3 min at 4K </li>
| + | |
| − | <li>wash out with MOPS and resuspend with 1 mL MOPS</li>
| + | |
| − | <li>repeat spin step and resuspend again </li>
| + | |
| − | <li>measure OD<sub>650</sub> of a 10 fold dilution of cells with a spectrophotometer blanked with MOPS </li>
| + | |
| − | <li>calculate the amount of inocula required for a final OD<sub>650</sub> of 0.4 in 500 uL</li>
| + | |
| − | <li>set up wells - 2 uL phosphate, 5 uL cells, 193 uL MOPS </li>
| + | |
| − | | + | |
| − | </ol>
| + | |
| − | | + | |
| | </div> | | </div> |
| | <div class="col-md-1"></div> | | <div class="col-md-1"></div> |
