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| | .he_top{position:absolute;left:15%;top:100px;width:70%;height:auto;font-family:Arial, Helvetica, sans-serif;font-size:50px;font-weight:bold;text-align:center;} | | .he_top{position:absolute;left:15%;top:100px;width:70%;height:auto;font-family:Arial, Helvetica, sans-serif;font-size:50px;font-weight:bold;text-align:center;} |
| Line 80: |
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| | <li>Add 1-2ul plasmid to 100ul competent cells. Homogenize by gently mixing with pipette several times. (Note that if the plasmid is product of linkage, add 10-20ul DNA)</li> | | <li>Add 1-2ul plasmid to 100ul competent cells. Homogenize by gently mixing with pipette several times. (Note that if the plasmid is product of linkage, add 10-20ul DNA)</li> |
| | <li>Keep on ice for 30min.</li> | | <li>Keep on ice for 30min.</li> |
| - | <li>Put it at 42℃ for 90s</li>
| |
| - | <li>Keep on ice again for 2min.</li>
| |
| - | <li>Add 400ul LB .</li>
| |
| - | <li>Incubate the cells at 37℃ with 100rpm for 45min.</li>
| |
| - | <li>Centriguged at 4000rpm for 1min.</li>
| |
| - | <li>Remove 300ul of supernatant.</li>
| |
| - | <li>Resuspended the rest of 200ul of cultures and plate it onto selective plates.</li>
| |
| - | </ul>
| |
| - | <br/><br/>
| |
| - | <p>
| |
| - | <span class="son">Eletrocompetent cell</span><br/>
| |
| - | <span class="title">Materials</span><br/>
| |
| - | LB medium,Spectrophotometer,Shaking incubator at 30℃,Ice-cold 10% glycerol,Icebox,-80℃ refrigerator
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="title">Protocol</span></p>
| |
| - | <ul>
| |
| - | <li>Inoculate 5ml LB with <i>E.coli</i>; incubate over night at 30℃ and 250 rpm. Our E.coli has transformed plasmid PKD46, which would lose at 37 ℃</li>
| |
| - | <li>Inoculate 100ml LB with an aliquot of an overnight culture and grow to an O.D.600 of 0.6 at 30℃ with 250 rpm. When the O.D.600 is 0.2, add 3mmol arabinose to induce the PKD46 to produce the protein we need</li>
| |
| - | <li>Divide the cultures into two 50ml Epppendorf tubes</li>
| |
| - | <li>Keep on ice for 30min</li>
| |
| - | <li>Centrifuged at 4000rpm for 4min at 4℃</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Resuspended in 10ml ice-cold sterile water</li>
| |
| - | <li>Keeping on ice for 10min</li>
| |
| - | <li>Centrifuged at 4000rpm for 4min at 4 ℃ again</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Resuspended in 10ml ice-cold 10% glycerol</li>
| |
| - | <li>Keeping on ice for 10min</li>
| |
| - | <li>Centrifuged at 4000rpm for 4min at 4 ℃ again</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Resuspended in 10ml ice-cold 10% glycerol</li>
| |
| - | <li>Keeping on ice for 10min</li>
| |
| - | <li>Repeat the step 9-12 again</li>
| |
| - | <li>Centrifuged at 4000rpm for 4min at 4 ℃</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Resuspended in 1ml 10% glycerol</li>
| |
| - | <li>Devide it into 1.5ml Eppendorf tubes, 100ul per tube</li>
| |
| - | <li>Stored at -80℃</li>
| |
| - | </ul>
| |
| - | <br/><br/>
| |
| - | <p>
| |
| - | <span class="son">Electrotransformation</span><br/>
| |
| - | <span class="title">Materials</span><br/>
| |
| - | 75% ethanol,Linear DNA fragement,SOC medium,Electroporation device,Icebox,Electrocompetent cell,Selective plate,Shaking incubator at 37 ℃,Eppendorf centrifugation
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="title">Protocol</span></p>
| |
| - | <ul>
| |
| - | <li>Taken from 75% ethanol, cuvette is exposed to ultraviolet ray for 30min in bechtop after drying</li>
| |
| - | <li>Then keep the sterilized cuvette on ice</li>
| |
| - | <li>Add 1-10ul(300ug) DNA to 100ul of electrocompetent cells</li>
| |
| - | <li>Homogenize by gently mixing with pipette several times and then transfer mixture into a prechilled cuvette</li>
| |
| - | <li>Wipe moisture from cuvette and insert the cuvette into the electroporation device</li>
| |
| - | <li>Electroporation</li>
| |
| - | </ul>
| |
| - |
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td class="name">Mode</td>
| |
| - | <td>Bacterial</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td class="name">Voltage</td>
| |
| - | <td>1.8kV</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td class="name">Time constant</td>
| |
| - | <td>6ms</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Immediately add 1ml of SOC medium</li>
| |
| - | <li>Transfer the mix into a sterile 1.5ml tube</li>
| |
| - | <li>Incubate at 37 ℃ with 150 rpm for 1h</li>
| |
| - | <li>Centriguged at 4000rpm for 1min</li>
| |
| - | <li>Discard 800ul of supernatant</li>
| |
| - | <li>Resuspend the rest of 200ul of culture solution and plate it onto selective plates</li>
| |
| - | </ul>
| |
| - | <br/><br/>
| |
| - | <p>
| |
| - | <span class="son">SDS-PAGE</span><br/>
| |
| - | <span class="title">Materials</span><br/>
| |
| - | Stacking gel,Separating gel,Comb,SDS running buffer,Gel electrophoresis,Collodial coomassie
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="title">Protocol</span><br/>
| |
| - | Pouring the polyacrylamide gel
| |
| - | </p>
| |
| - | <ul>
| |
| - | <li>Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED</li>
| |
| - | <li>Aliquote 6.5 mL for each separating and 2.5 mL for each stacking gel</li>
| |
| - | <li>Add ammonium persulfate and TEMED to each separating gel aliquote</li>
| |
| - | <li>Pour the solution quickly into the gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel</li>
| |
| - | <li>Layer wateter on top of the gel</li>
| |
| - | <li>Leave the separating gel at room temperature for 1h to polymerize</li>
| |
| - | <li>Remove water and wait until the surface is dry</li>
| |
| - | <li>Add ammonium persulfate and TEMED to each stacking gel aliquote and pour the solution quickly into your gel casting form</li>
| |
| - | <li>Insert comb without getting bubbles stuck underneath</li>
| |
| - | <li>Leave the gel at room temperature for 1h to polymerize</li>
| |
| - | <li>Remove sealing and store the gel wrapped in moistened paper towel at 4°C</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Running the gel</p>
| |
| - | <ul>
| |
| - | <li>Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber</li>
| |
| - | <li>Remove comp without destroying the gel pocket</li>
| |
| - | <li>Pipet the sample into the gel pockets</li>
| |
| - | <li>Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel</li>
| |
| - | <li>Raise amperage up to 20 mA for running the separating gel</li>
| |
| - | <li>When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Collodial coomassie staining</p>
| |
| - | <ul>
| |
| - | <li>Agitate the staining solution at 37 °C over night to form the colloids</li>
| |
| - | <li>After finishing the SDS-PAGE remove gel from gel casting form and transfer it in to a box</li>
| |
| - | <li>Add 100 mL of the staining solution to your polyacrylamid gel</li>
| |
| - | <li>Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation</li>
| |
| - | <li>Remove the staining solution</li>
| |
| - | <li>Wash the gel with ddH<sub>2</sub>O</li>
| |
| - | <li>Incubate the gel in ddH<sub>2</sub>O (2-6 h) for bleaching the background</li>
| |
| - | <li>Shake the gel continuously during incubation. If necessary replace the colored water with new one</li>
| |
| - | </ul>
| |
| - | <br/><br/>
| |
| - | <p>
| |
| - | <span class="son">Western blot</span><br/>
| |
| - | <span class="title">Materials</span><br/>
| |
| - | PBS
| |
| - | Eppendorf centrifugation,Protease inhibitor,Spectrophotometer,Sample buffer,Electrophorator,Sponge,PDVF,Methanol
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="title">Protocol</span><br/>
| |
| - | Protein extraction from adherent cells
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS</li>
| |
| - | <li>Add PBS and use a cell scraper to dislodge the cells. Move the mixture into microcentrifuge tubes</li>
| |
| - | <li>Centrifuge at 1500 RPM for 5 minutes</li>
| |
| - | <li>Discard the supernatant</li>
| |
| - | <li>Add 180 μl of ice cold cell lysis buffer with 20 μl fresh protease inhibitor cocktail. (Make sure the protein concentration is high enough, if not it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail)</li>
| |
| - | <li>Incubate for 30 minutes on ice</li>
| |
| - | <li>Clarify the lysate by spinning for 10 minutes at 12000 RPM at 4℃</li>
| |
| - | <li>Transfer supernatant (or protein mix) to a fresh tube</li>
| |
| - | <li>Measure the concentration of protein using a spectrophotometer</li>
| |
| - | <li>store on ice or frozen at -20℃or -80 ℃</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Sample preparation</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Put 50μg of protein extract in each well</li>
| |
| - | <li>Add 5 μl sample buffer to the sample, and make the volume in each lane equalized using ddH<sub>2</sub>O. Mix the buffer and the sample</li>
| |
| - | <li>Heat the samples with dry plate at 100℃ for 5 minutes</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Electrophoresis</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Pour the running buffer into the electrophorator</li>
| |
| - | <li>Place the gel inside the electrophorator and connect to a power supply</li>
| |
| - | <li>Make sure the buffer covers the gel completely, and remove the comb</li>
| |
| - | <li>Load 6μl of marker and 15 μl of samples into each well</li>
| |
| - | <li>Run the gel with low voltage (60 V) for separating gel ; higher voltage (140 V) for stacking gel</li>
| |
| - | <li>Run the gel around an hour, or until the dye front runs off the bottom of the gel</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Electrotransfer</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Cut 6 filter sheets to fit the measurement of the gel, and one polyvinylidene fluoride (PDVF) membrane with the same dimensions</li>
| |
| - | <li>Wet the sponge and filter paper in transfer buffer, and wet the PDVF membrane in methanol</li>
| |
| - | <li>Separate glass plates and retrieve the gel</li>
| |
| - | <li>Create a transfer sandwich as follows: Sponge, 3 Filter Papers, Gel PVDF, 3 Filter Papers.(Make sure there is no air bubbles between the gel and PVDF membrane)</li>
| |
| - | <li>Relocate the sandwich to the transfer apparatus, which should be placed on ice to maintain 4℃</li>
| |
| - | <li>Add transfer buffer to the apparatus, and ensure that the sandwich is covered with the buffer</li>
| |
| - | <li>Place electrodes on top of the sandwich, ensuring that the PVDF membrane is between the gel and a positive electrode</li>
| |
| - | <li>Transfer for 90 minutes</li>
| |
| - | </ul>
| |
| - | <br/><br/>
| |
| - | <p>
| |
| - | <span class="son">RNA extraction</span><br/>
| |
| - | <span class="title">Materials</span><br/>
| |
| - | water bath 65℃,AE,SDS,Phenol,Chloroform,Eppendorf centrifugation,RNase free water,75% ethanol,Absolute ethanol
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="title">Protocol</span></p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the 450ul AE,50ul SDS,350ul phenol and 350ul chloroform</li>
| |
| - | <li>Pre-heated at 65 ℃</li>
| |
| - | <li>Add 1ml the mixed liquor to resuspent the pelleted cells</li>
| |
| - | <li>Shake violently</li>
| |
| - | <li>Put it in 65 ℃ water bath evaportor</li>
| |
| - | <li>Keep on ice for 5min</li>
| |
| - | <li>Centrifuged with 1200g for 7min at 4 ℃</li>
| |
| - | <li>The supernatant is transferred to a 1.5ml Eppendorf tube</li>
| |
| - | <li>Mix with 600ul chloroform</li>
| |
| - | <li>Centrifuged with 1200g for 7min at 4 ℃</li>
| |
| - | <li>The supernatant is transferred to a new Eppendorf tube</li>
| |
| - | <li>Add 1/10 volume NaAc and 2.5 times the volume absolute ethanol</li>
| |
| - | <li>Keep at -20℃ for 30min</li>
| |
| - | <li>Centrifuged with 1200g for 15min at 4 ℃</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Resuspended with 1ml 75% ethanol</li>
| |
| - | <li>Centrifuged with 1200g for 5min at 4 ℃</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>The tube is exposed at in bechtop for 2-3min to remove the liquid totally</li>
| |
| - | <li>Add 20ul RNase free water to dissolve the RNA sediment</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>
| |
| - | <span class="son">PCR</span><br/><br/>
| |
| - | <span class="title">Real time PCR</span><br/>
| |
| - | Remove genome DNA
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Prepare 10ul reaction in 0.5ml PCR tube</li>
| |
| - | </ul>
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td class="name">Ingredient</td>
| |
| - | <td class="name">Amount(ul)</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>5×gDNA Eraser Buffer</td>
| |
| - | <td>2</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>gDNA Eraser</td>
| |
| - | <td>1</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Total RNA</td>
| |
| - | <td>*1</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>RNase Free dH2O</td>
| |
| - | <td>Up to 10</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the reaction and spin down in microcentrifuge</li>
| |
| - | <li>Put the tube in 42 ℃ water bath for 2min</li>
| |
| - | <li>Stored in 4 ℃</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Reverse transcription</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Prepare 20ul reaction in 0.5ml PCR tube</li>
| |
| - | </ul>
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td class="name">Ingredient</td>
| |
| - | <td class="name">Amount(ul)</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>The above mixture</td>
| |
| - | <td>10</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>PrimeScript RT Enzyme Mix I</td>
| |
| - | <td>1</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>RT Primer Mix</td>
| |
| - | <td>1</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>5×PrimeScript Buffer 2(for Real Time)</td>
| |
| - | <td>4</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>RNase Free ddH<sub>2</sub>O</td>
| |
| - | <td>4</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Total</td>
| |
| - | <td>20</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the reaction and spin down in microcentrifuge</li>
| |
| - | <li>Put the tube in 37 ℃ water bath for 15min</li>
| |
| - | <li>Then transfer the tube in 85 ℃ water bath for 5sec</li>
| |
| - | <li>Stored in 4 ℃</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Real time PCR</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Prepare 25ul reaction in 0.5ml PCR tube</li>
| |
| - | </ul>
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td class="name">Ingredient</td>
| |
| - | <td class="name">Amount(ul)</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>SYBR Premix Ex TaqII(Tli RNaseH Plus)(2×)</td>
| |
| - | <td>12.5</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Forward Primer(10uM)</td>
| |
| - | <td>1.0</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Reverse Primer(10uM)</td>
| |
| - | <td>1.0</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>RT reaction</td>
| |
| - | <td>2</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>dH2O</td>
| |
| - | <td>8.5</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Total</td>
| |
| - | <td>25</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the reaction and spin down in microcentrifuge</li>
| |
| - | <li>Run the PCR machine using bellowing conditions</li>
| |
| - | </ul>
| |
| - |
| |
| - | <img src="https://static.igem.org/mediawiki/2014/2/21/Num2.png" style="width:250px;">
| |
| - |
| |
| - | <p><span class="title">Taq PCR</span></p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Prepare 20ul reaction in 0.5ml PCR tube</li>
| |
| - | </ul>
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td class="name">Ingredient</td>
| |
| - | <td class="name">Amount(ul)</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>λDNA(2.5ng/ul)</td>
| |
| - | <td>1</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Forward Primer(10uM)</td>
| |
| - | <td>2</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Reverse Primer(10uM)</td>
| |
| - | <td>2</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>2×PCR Mix</td>
| |
| - | <td>25</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>ddH2O</td>
| |
| - | <td>Up to 20</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the reaction and spin down in microcentrifuge</li>
| |
| - | <li>Run the PCR machine using bellowing conditions</li>
| |
| - | </ul>
| |
| - |
| |
| - | <img src="https://static.igem.org/mediawiki/2014/d/d5/Num3.png" style="width:250px;">
| |
| - | <br/>
| |
| - | <p><span class="title">KOD PCR</span>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Prepare 50ul reaction in 0.5ml PCR tube</li>
| |
| - | </ul>
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td class="name">Ingredient</td>
| |
| - | <td class="name">Amount(ul)</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>dNTPs</td>
| |
| - | <td>10</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>2×PCR buffer for KOD FX</td>
| |
| - | <td>25</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Forward Primer(10uM)</td>
| |
| - | <td>1.5</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Reverse Primer(10uM)</td>
| |
| - | <td>1.5</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>KOD FX enzyme</td>
| |
| - | <td>1</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Genome(plasmid orλDNA)</td>
| |
| - | <td>200ng(50ng or 50ng)</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>ddH2O</td>
| |
| - | <td>up to 50</td>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the reaction and spin down in microcentrifuge</li>
| |
| - | <li>Run the PCR machine using bellowing conditions</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p><img src="https://static.igem.org/mediawiki/2014/4/42/Num1.png" style="width:250px;"></p>
| |
| - | <br/><br/>
| |
| - |
| |
| - | <p><span class="son">GC-MS</span><br/>
| |
| - | <span class="title">Materials</span><br/>
| |
| - | Ethanol,Chloroform,Water bath at 80 ℃,NaOH,Methanol,NaCl,GC-MS
| |
| - |
| |
| - | <p><span class="title">Protocol</span><br/>
| |
| - | Sample Preparation
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Add 2ml ethanol and 1ml chloroform to a flask</li>
| |
| - | <li>Add 2 drops of 6M HCl into the flask</li>
| |
| - | <li>Refluxed at 80 ± 2℃ in water bath with stirring for 30 minutes</li>
| |
| - | <li>After completing the reflux, extract with 1 ml of the mixture of diethyl ether and petroleum ether (1:1) for two times with a separatory funnel</li>
| |
| - | <li>Collect the organic layer in the separatory funnel after every extraction</li>
| |
| - | <li>Combined them into another round bottom flask, and evaporate in a water bath with stirring until near to dryness</li>
| |
| - | <li>Add 2 ml of 0.5 M NaOH in methanol into the round bottom flask</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Derivatisation</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Transfer 1ml of extract to another a screw cap test tube</li>
| |
| - | <li>Add 1 ml of BF3</li>
| |
| - | <li>The solution is refluxed for an additional 5 minutes</li>
| |
| - | <li>After cooling down, add 1ml of the mixture of 0.5 μl/ml 1-bromotetradecane (the internal standard) in n-heptane and 1 ml of saturated sodium chloride solution</li>
| |
| - | <li>Gently shake the screw cap test tube for several times and collect the upper layer into a vial for GC analysis</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>GC Analysis</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>1μl of sample solution is injected into GC. Make sure the GC is set using the following condition</li>
| |
| - | <li>Set the automatic run for your samples using the autosampler</li>
| |
| - | <li>Record the peak area and retention time of each analytical peak for calculation</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>
| |
| - | <span class="son">Immunodetection</span><Br/><br/>
| |
| - | <span class="title">Materials</span><br/>
| |
| - | Alexa Floor 488,Fluorescence microscope,Shaking incubator at 37℃,1×PBS,Microscope slide,1% BSA
| |
| - |
| |
| - | <p><span class="title">Protocol</span>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Take out 1ml E.coli which have been induced with IPTG</li>
| |
| - | <li>Centrifuged with the speed of 6000g for 5 min</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Add 1×PBS to resuspend the thallus</li>
| |
| - | <li>Centrifuged with 6000g, 5 min</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Add 1×PBS to resuspend the thallus</li>
| |
| - | <li>Wash the thallus with 1×PBS like above for 3 times</li>
| |
| - | <li>Add optimum amount TBS containing 1% BSA to resuspend the thallus and adjust the OD<sub>600</sub> to 5</li>
| |
| - | <li>Add 1.2ul monoclonal antibody to 200ul of cell suspension</li>
| |
| - | <li>Incubate the mix at 37℃ for 2h</li>
| |
| - | <li>Centrifuged the mix with 6000g for 5min</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Add 1×PBS to resuspend the thallus</li>
| |
| - | <li>Wash the thallus with 1×PBS like above for 3 times</li>
| |
| - | <li>resuspended with 1×PBS containing 1% BSA</li>
| |
| - | <li>Centrifuged the mix with 6000g for 5min</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Resuspended with 1×PBS containing 1% BSA adding 1.2 ul Alexa Floor 488 which is an IgG antibody</li>
| |
| - | <li>Incubate the mix at 37℃ for 1h</li>
| |
| - | <li>Add 1×PBS to resuspend the thallus</li>
| |
| - | <li>Discard supernatant</li>
| |
| - | <li>Add 1×PBS to resuspend the thallus</li>
| |
| - | <li>Wash the thallus with 1×PBS like above for 3 times</li>
| |
| - | <li>Resuspended with 1.5ml 1×PBS</li>
| |
| - | <li>Take out 2ul cell suspension to put on microscope slide and then observe it by fluorescence microscope</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>
| |
| - | <span class="son">Digestion</span><br/><br/>
| |
| - | <span class="title">Materials</span><br/><br/>
| |
| - | plasmid DNA or PCR product,restriction enzymes,buffer (10x),ddH<sub>2</sub>O,water bath at 37 °C,heat block or water bath at 65 °C
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="title">Protocol</span></p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Prepare 20ul reaction in 0.5ml PCR tube</li>
| |
| - | </ul>
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td>ddH2O</td>
| |
| - | <td>15ul</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>10×FastDigest Green buffer</td>
| |
| - | <td>2ul</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Plasmid(PCR product or genome)</td>
| |
| - | <td>2ul(up to 1ug) or 10ul(~0.2ug) or 10ul(5ug)</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>FastDigest enzyme</td>
| |
| - | <td>1ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the reaction and spin down in microcentrifuge</li>
| |
| - | <li>Incubate for (at least) one hour at 37 °C</li>
| |
| - | <li>Inactivate the restriction endonucleases by heat, incubation at 65 °C for 10 minutes and centrifuge shortly</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>
| |
| - | <span class="son">Ligation</span><br/><br/>
| |
| - | <span class="title">Materials</span><br/><br/>
| |
| - | digested plasmid DNA or PCR product,T4 ligation buffer (10x),T4 ligase ,H2O,water bath at 16 °C
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="son">Protocol</span></p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Prepare 10ul reaction in 0.5ml PCR tube</li>
| |
| - | </ul>
| |
| - |
| |
| - | <table>
| |
| - | <tr>
| |
| - | <td>10×T4 buffer</td>
| |
| - | <td>2.5ul</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>T4 enzyme</td>
| |
| - | <td>1ul</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>DNA inserter</td>
| |
| - | <td>10 times to DNA vector</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>DNA vector</td>
| |
| - | <td>1/10 times to DNA inserter</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>ddH2O</td>
| |
| - | <td>Add to 25ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Mix the reaction and spin down in microcentrifuge</li>
| |
| - | <li>Put it in16 °C water bath overnight</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>
| |
| - | <span class="son">Gel running</span><br/><br/>
| |
| - | <span class="title">Materials</span><br/><br/>
| |
| - | Agarose ,TAE 1x ,SybrSafe DNA stain,Loading Dye,DNA ladder (Smartladder),DNA electrophoresis machine
| |
| - |
| |
| - | <span class="title">Protocol</span>
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Dissolve agarose (w/v 0.6% for separating long DNA pieces (>10 kbp), 1% for separating shorter pieces) in 1x TAE by microwaving</li>
| |
| - | <li>Close sides of electrophoresis tray (scotch tape works fine) and add comb</li>
| |
| - | <li>Let solution cool and add 5 µl Sybrsafe to an empty electrophoresis tray (small gels) 10-12 µl Sybrsafe for larger gels</li>
| |
| - | <li>Pour gel until a height of ~0.5 cm. Mix and remove bubbles with pipet tip (fast! It hardens quickly)</li>
| |
| - | <li>Put tray into electrophoresis casing and add TAE until a small layer above the gel can be seen. Remove comb</li>
| |
| - | <li>Add 1 µl loading dye to 5 µl sample, mix and load in the gel. Also add 5 µl smartladder for your reference</li>
| |
| - | <li>Run gel on 80 V (long run)- 110 V (short run, mostly for a 'fast check') for ~40-60 minutes, dependant of gel size, separation acquisition and voltage</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>
| |
| - | <span class="son">Material</span><br/><br/>
| |
| - | <span class="title">LB medium</span><Br/><br/>
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>10 g Trypton</li>
| |
| - | <li>5 g yeast extract</li>
| |
| - | <li>10 g NaCl</li>
| |
| - | <li>12 g Agar-Agar (for plates)</li>
| |
| - | <li>Adjust pH to 7.4</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p><span class="title">SOC medium</span><br/>
| |
| - | Add the following components for 900 ml of distilled ddH<sub>2</sub>O:
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>20 g Trypton</li>
| |
| - | <li>5 g Bacto Yeast Extract</li>
| |
| - | <li>2 mL of 5 M NaCl</li>
| |
| - | <li>2.5 ml of 1 M KCl</li>
| |
| - | <li>10 ml of 1 M MgCl<sub>2</sub></li>
| |
| - | <li>10 ml of 1 M MgSO<sub>4</sub></li>
| |
| - | <li>20 ml of 1 M glucose</li>
| |
| - | </ul>
| |
| - | <p>Adjust to 1L with distilled H<sub>2</sub>O. Sterilize by autoclaving</p>
| |
| - |
| |
| - | <p><span class="title">TAE-Buffer</span><br/>
| |
| - | 1 L of 50x TAE buffer
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>242.48 g Tris</li>
| |
| - | <li>41.02 g sodium acetate</li>
| |
| - | <li>18.612 g EDTA</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>
| |
| - | Adjust pH to 7.8 with acetic acid<br/>
| |
| - | Solve in ddH<sub>2</sub>O<br/>
| |
| - | Dilute 20 mL 50x stock in 1L dH2O for 1x Buffer for PAGE<br/>
| |
| - | </p>
| |
| - |
| |
| - | <p><span class="title">SDS-PAGE gel</span><Br/>
| |
| - | Stacking gel 5 %:
| |
| - | </p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>775 μL ddH<sub>2</sub>O</li>
| |
| - | <li>1.25 mL 0,25 M Tris (pH 6,8)</li>
| |
| - | <li>425 μL Bis/Acrylamide (0,8 %, 30 %)</li>
| |
| - | <li>50 μL 5 % SDS</li>
| |
| - | <li>25 μL 10 % Ammonium persulfate</li>
| |
| - | <li>3 μL TEMED</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Separating gel 12 %:</p>
| |
| - | <ul>
| |
| - | <li>1.5 mL ddH<sub>2</sub>O</li>
| |
| - | <li>2.8 mL 1 M Tris (pH 8,8)</li>
| |
| - | <li>3.0 mL Bis/ Acrylamide (0,8%, 30%)</li>
| |
| - | <li>150 μL 5% SDS</li>
| |
| - | <li>37.5 μL 10% Ammonium persulfate</li>
| |
| - | <li>5 μL TEMED</li>
| |
| - | </ul>
| |
| - |
| |
| - |
| |
| - |
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| - | </div> <!--words over-->
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| - |
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| - |
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| - |
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| - | </body>
| |
| - | </html>
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