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− | {{ASTWS-China}}
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| + | <div class="vlogo"><img src="https://2017.igem.org/wiki/skins/Igem/images/IGEM_white_letters.png" alt="" /></div> |
| + | <ul id="options" class="clearfix"> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China">HOME</a> |
| + | </li> |
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| + | <a class="sub-nav-toggle">TEAM</a> |
| + | <ul class="sub-nav hidden"> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/AboutUs">About Us</a> |
| + | </li> |
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| + | <a href="https://2017.igem.org/Team:ASTWS-China/Members">Members</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Collaborations">Collaborations</a> |
| + | </li> |
| + | </ul> |
| + | </li> |
| + | <li> |
| + | <a class="sub-nav-toggle">PROJECT</a> |
| + | <ul class="sub-nav hidden"> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Description" class="selected">Description</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Design">Design</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Experiments">Experiment</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Notebook">Notebook</a> |
| + | </li> |
| | | |
− | <div class="column full_size"> | + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/InterLab">InterLab</a> |
| + | </li> |
| | | |
− | <h1>Notebook</h1> | + | <li> |
− | <p> Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.</p> | + | <a href="https://2017.igem.org/Team:ASTWS-China/Contribution">Contribution</a> |
| + | </li> |
| | | |
− | </div> | + | <li> |
− | <div class="clear"></div> | + | <a href="https://2017.igem.org/Team:ASTWS-China/Model">Model</a> |
| + | </li> |
| | | |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Results">Results</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Demonstrate">Demonstrate</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Improve">Improve</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Attributions">Attributions</a> |
| + | </li> |
| + | </ul> |
| + | </li> |
| + | <li> |
| + | <a class="sub-nav-toggle">PARTS</a> |
| + | <ul class="sub-nav hidden"> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Parts">Parts</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Basic_Part" >Basic Parts</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Composite_Part">Composite parts</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Part_Collection">Part Collection</a> |
| + | </li> |
| + | </ul> |
| + | </li> |
| + | <li> |
| + | <a class="sub-nav-toggle">HUMAN PRACTICE</a> |
| + | <ul class="sub-nav hidden"> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/HP/Silver" class="selected">Silver</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/HP/Gold_Integrated">Integrated and Gold</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Engagement">Public Engagement</a> |
| + | </li> |
| + | </ul> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Applied_Design">Applied Design</a> |
| + | </li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China/Safety">SAFETY</a> |
| + | </li> |
| + | </ul> |
| + | </nav> |
| | | |
− | <div class="column half_size"> | + | <div id="wrap"> |
− | <h5>What should this page have?</h5> | + | <div class="content-wrapper"> |
− | <ul>
| + | |
− | <li>Chronological notes of what your team is doing.</li>
| + | |
− | <li> Brief descriptions of daily important events.</li>
| + | |
− | <li>Pictures of your progress. </li>
| + | |
− | <li>Mention who participated in what task.</li>
| + | |
− | </ul>
| + | |
| | | |
− | </div> | + | <header id="header" class="clearfix"> |
| + | <div class="logo-wrapper"> |
| + | <h1 id="logo"> |
| + | <a href="https://2017.igem.org/Team:ASTWS-China">ASTWS-China</a> |
| + | </h1> |
| + | </div> |
| + | <div id="menu-button"> |
| + | <div class="centralizer"> |
| + | <div class="cursor"> |
| + | <div id="nav-button"> |
| + | <span class="nav-bar"></span> |
| + | <span class="nav-bar"></span> |
| + | <span class="nav-bar"></span> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </header> |
| | | |
− | <div class="column half_size"> | + | <div id="content"> |
− | <h5>Inspiration</h5> | + | <div id="container" class="clearfix"> |
− | <p>You can see what others teams have done to organize their notes:</p>
| + | |
| | | |
− | <ul> | + | <article class="element clearfix col3-3 home auto"> |
− | <li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li> | + | <div class="boxed"> |
− | <li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li> | + | <h2 class="header">NOTEBOOK</h2> |
− | <li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li> | + | <p> |
− | <li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li> | + | <br/> |
− | </ul> | + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 18px">Agarose Gel Electrophoresis</span></strong> |
| + | </p> |
| + | <p style="text-align: left; line-height: 150%;"> |
| + | <span style="font-family: "Times New Roman"; font-size: 14px;">1. Weigh agarose powder and TAE buffer according to a proper portion, and add them to a 100ml conical flask (we usually make 1.5% Agarose Gel).</span><br/> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: "Times New Roman"; line-height: 150%; font-size: 14px;">3. Let the solution cool down to about 40-50</span><span style="font-family: "Times New Roman"; line-height: 150%; font-size: 14px;">℃</span><span style="font-family: "Times New Roman"; line-height: 150%; font-size: 14px;"> and add DNA gel stain (usually we use EB), pour the solution into the gel casting tray with appropriate comb. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. Let the gel cool until it becomes solid.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Pull out the comb carefully.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6. Place the gel in the electrophoresis chamber.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7. Add enough TAE Buffer so that there is about 2-3mm of buffer over the gel.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: "Times New Roman"; font-size: 14px;">8. Pipette DNA samples mixed with appropriate amount of DNA loading buffer (the dye/GeneFinder is in the loading buffer) into wells on the gel.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">9. Run the gel at 135V for about twenty minutes.</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Basic ELISA Protocol</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">There are many different types of ELISAs, which can detect the presence of protein in serum or </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">supernatent. One of the most common types of ELISA is the so-called "sandwich ELISA." It is termed this because the antibody that you are detecting gets sandwiched between an antigen and a chromogenically-conjugated antibody. Below you will find a basic protocol for this assay.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">To coat the plate with the appropriate antigen, fill the microwells of a Immuno Plate with 100uL of the diluted antigen.</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">2<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Incubate at 4</span></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">℃</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">overnight</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Wash the unbound antigen off the plate by flicking the contents of the plate into the sink, fill the wells with PBS-Triton, flick again, repeat 2X with PBS-Triton.</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Block non-specific binding by adding 200uL of 1%BSA/PBS</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Incubate for 30-60minutes at Room Temperature (RT)</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">6<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Wash plate as above</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">7<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Add 100uL of (diluted) samples to appropriate wells. Be sure to include positive and negative controls, and, if necessary, a standard curve.</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">8<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Incubate for 1hour at RT</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">9<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Repeat washing step</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">10<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Prepare appropriate dilution of the second step antibody conjugated either with Alkaline Phosphatase or Horseradish Peroxidase. (antibodies should be titrated for optimal dilution)</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">11<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Add 100uL of second step antibody to wells and incubate for 1hr</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">12<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Repeat washing step</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">13<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Prepare substrate solution*</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">14<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Add 100uL of substrate to well and incubate at RT for 60min</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">15<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">add stopping solution if appropriate</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">16<span style="font-family:微软雅黑">、</span><span style="font-family:Times New Roman">Read plates on an ELISA plate reader</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">*Common Substrates and the appropriate plate reader setting</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">ABTS: 405-410 nm </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">TMB: non-stopped 620-650 nm, stopped 450 nm </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">OPD: non-stopped 450 nm, stopped 490 nm </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">pNPP: 405-410 nm </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">BluePhosTM: 595-650 nm </span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Bind with NTA-Ni beads</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1.</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(238,240,242)"> To harvest overnight induced E. coli (OD: 4.0)200ul. 4000rpm centrifuge 1min and remove the upper cleaning fluid.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">Use PBS to clean the bacteria in the EP tube twice, and use 250ul PBS to resuspent the bacteria.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Cells were fixed by mixing 250 ul cells with a 750 u1 4 % (w/v)solution of paraformaldehyde in PBS. This mixture was incubated on ice for 20 min.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. To remove the fixative, cells were washed twice in PBS. </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">and use 250ul PBS to resuspent the bacteria.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">Adding</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">primary</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">antibody</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">(monoclonal) ,1:2000 dilution, at 4</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">incubation for one</span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)"> hours</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6. Cells were washed twice in PBS. </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">and use 250ul PBS to resuspent the bacteria.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7.</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> Adding</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">second antibody(</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">FITC-conjugated</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">)</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">,</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1: 5 000 dilution, at 4</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> incubation for one </span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)">hours</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">8. </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">Use PBS to clean the bacteria in the EP tube twice, and use 250ul PBS to resuspent the bacteria.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">9.</span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)"> Adding 100ul NTA-Ni (50% slurry) to EP tube,</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)">NTA-Ni </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> were washed twice in PBS.</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)"> and use 50ul PBS to resuspent the </span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)">NTA-Ni</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">10.</span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)"> The NTA-Ni and </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)"> bacteria cells</span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)"> avoided the light for </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">incubation</span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(238,240,242)"> two hours.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(238, 240, 242)">11. The NTA-Ni and </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> bacteria</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> cells were washed twice in PBS,</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> and use 250ul PBS to resuspended.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">12. Gravity naturally subsidence for half an hour, sent to</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">microscopic</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">examination for </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> </span><a href="http://undefined"><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(0,0,0);font-size:14px;background:rgb(249,251,252)">precipitate</span></a><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">.</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">cation- exchange chromatography</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Sample preparation</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">For optimal growth, induction and cell lysis conditions, please refer to established protocols.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">The sample should be fully dissolved. To avoid column clogging, we recommend centrifugation and filtration through a 0.22 μm filter to remove cell debris or other particulate material. </span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">Purification procedure for a packed column </span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">1. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water. </span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">2. Equilibrate the column with 5</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">-10</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)"> </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">column volumes of binding buffer.</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> or until the column effluent shows stable conductivity and pH values.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Bind the sample</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1. Add 10 ml of sample to 1 ml of the 50% slurry. Binding capacity of SP Sepharose Fast Flow</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> is protein dependent and the average is 40 mg/ml. This means that 1 ml of the 50% slurry can bind about 20 mg of total protein.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">2. Incubate sample and the SP Sepharose Fast Flow slurry at room temperature on a shaker at low speed for 1 h.</span> |
| + | </p> |
| + | <p style="text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">3. Load sample/SP Sepharose Fast Flow mix onto the 1ML columns and collect flowthrough.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Buffer wash and elution</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1. Wash with 5 × 1 ml of binding buffer and collect both fractions.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">2. Elute with 4×1ml of elution buffer and collect the eluted fractions in four separate tubes.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Regeneration </span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">After each separation, elute any reversibly bound material either with a high ionic strength solution(1 M NaCl in buffer) or by increasing pH. Regenerate the medium by washing with at least 5 bed volumes of buffer, or until the column effluent shows stable conductivity and pH values.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Measure absorbance at 280 nm using a spectrophotometer and confirm purity of pooled fractions by SDS-PAGE. Use elution buffer as the blank.</span> |
| + | </p> |
| + | <p style="margin-bottom:8px"> |
| + | <strong><span style="font-family: 'Times New Roman';font-size: 14px">Store </span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:8px"> |
| + | <span style="font-family: 'Times New Roman';font-size: 14px">Store the beads in 20% EtOH.</span> |
| + | </p> |
| + | <table style="width: 558px;margin-left: 9px;margin-right: 9px"> |
| + | <tbody> |
| + | <tr style="height:37px"> |
| + | <td width="167" valign="top" style="width: 168px;padding: 0 7px;border-width: 1px;border-color: windowtext"> |
| + | <p style="margin-bottom:8px;text-align:center;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Wash/Bind Buffer (25 ml)</span> |
| + | </p> |
| + | </td> |
| + | <td width="189" valign="top" style="width: 189px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top-width: 1px;border-top-color: windowtext;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;text-indent:21px;text-align:center;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Elution Buffer (10 ml)</span> |
| + | </p> |
| + | </td> |
| + | <td width="200" valign="top" style="width: 201px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top-width: 1px;border-top-color: windowtext;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;text-indent:14px;text-align:center;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">Regeneration</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> Buffer (10 ml)</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr style="height:94px"> |
| + | <td width="167" valign="top" style="width: 168px;padding: 0 7px;border-left-width: 1px;border-left-color: windowtext;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">50 mM Tris-HCI pH 6.4</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Adjust pH to 6.4 w/HCI</span> |
| + | </p> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | </td> |
| + | <td width="189" valign="top" style="width: 189px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">50 mM Tris-HCI pH 7.5</span> |
| + | </p> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">500 mM NaCl</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Adjust pH to 7.5 w/ HCI</span> |
| + | </p> |
| + | </td> |
| + | <td width="200" valign="top" style="width: 201px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">50 mM Tris-HCI pH 6.0</span> |
| + | </p> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1 M NaCl</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Adjust pH to 6.0 w/ HCI</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">reagent</span></strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span> |
| + | </p> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Ni affinity chromatography</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Sample preparation</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">For optimal growth, induction and cell lysis conditions, please refer to established protocols.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">The sample should be fully dissolved. To avoid column clogging, we recommend centrifugation and filtration through a 0.22 μm filter to remove cell debris or other particulate material. </span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">Purification procedure for a packed column </span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">1. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water. </span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">2. Equilibrate the column with 5</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">-10</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)"> </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">column volumes of binding buffer.</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> or until the column effluent shows stable conductivity and pH values.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)">3.To prevent binding of host cell proteins with exposed histidine, add the same concentration of imidazole to the sample as to the binding buffer.</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(252, 252, 252)"> </span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Bind the sample</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1. Add 5 ml of sample to 1 ml of the 50% slurry. Binding capacity of </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> Ni Sepharose 6 Fast Flow</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> is protein dependent and the average is 20 mg/ml. This means that 1 ml of the 50% slurry can bind about 10 mg of target protein.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">2. Incubate sample and the </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Ni Sepharose 6 Fast Flow</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> slurry at room temperature on a shaker </span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">at low speed for 1 h.</span> |
| + | </p> |
| + | <p style="text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">3. Load sample/</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Ni Sepharose 6 Fast Flow</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> mix onto the 1ML columns and collect flowthrough.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Buffer wash and elution</span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1. Wash with 5 × 1 ml of binding buffer and collect both fractions.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">2. Elute with 4×1ml of elution buffer and collect the eluted fractions in four separate tubes.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Regeneration </span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1.After each separation, Regenerate the medium by washing with at least 5 bed volumes of buffer, or until the column effluent shows stable conductivity and pH values.</span> |
| + | </p> |
| + | <p style="margin-bottom:0;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">2.Measure absorbance at 280 nm using a spectrophotometer and confirm purity of pooled fractions by SDS-PAGE. Use elution buffer as the blank.</span> |
| + | </p> |
| + | <p style="margin-bottom:8px"> |
| + | <strong><span style="font-family: 'Times New Roman';font-size: 14px">Store </span></strong><strong></strong> |
| + | </p> |
| + | <p style="margin-bottom:8px"> |
| + | <span style="font-family: 'Times New Roman';font-size: 14px">Store the beads in 20% EtOH.</span> |
| + | </p> |
| + | <table style="width: 633px;margin-left: 9px;margin-right: 9px"> |
| + | <tbody> |
| + | <tr style="height:37px"> |
| + | <td width="205" valign="top" style="width: 206px;padding: 0 7px;border-width: 1px;border-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">6XHis Wash Buffer (25 ml)</span> |
| + | </p> |
| + | </td> |
| + | <td width="200" valign="top" style="width: 201px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top-width: 1px;border-top-color: windowtext;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">6X His Elution Buffer (10 ml)</span> |
| + | </p> |
| + | </td> |
| + | <td width="226" valign="top" style="width: 227px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top-width: 1px;border-top-color: windowtext;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">6X His </span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">Regeneration</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> Buffer (10 ml)</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr style="height:132px"> |
| + | <td width="205" valign="top" style="width: 206px;padding: 0 7px;border-left-width: 1px;border-left-color: windowtext;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">50 mM Phosphate Buffer pH 7.0</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">300 mM NaCl</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1 mM Imidizole</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Adjust pH to 7.0 w/NaOH</span> |
| + | </p> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | </td> |
| + | <td width="200" valign="top" style="width: 201px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">50 mM Phosphate Buffer pH 7.0</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">300 mM NaCl</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">300 mM Imidizole</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Adjust pH to 7.0 w/NaOH</span> |
| + | </p> |
| + | </td> |
| + | <td width="226" valign="top" style="width: 227px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">50 mM Phosphate Buffer pH 7.0</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1 M NaCl</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1 M Imidizole</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Adjust pH to 7.0 w/NaOH</span> |
| + | </p> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> reagent</span></strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"><br/></span> |
| + | </p> |
| + | <p style="margin-bottom:8px;line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">DNA purification/Axygen gel extraction</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. Absorb the liquids left on the surface of the gel slices using paper towels. Weigh gel slice (tare with empty tube).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Add 3 volumes of DE-A buffer per mg of gel (so a 100mg gel gets 300ul of buffer).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> until the gel is completely dissolved (keep heating for 6-8 minutes). If low-melt agarose gel is used, you may heat it at 40</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">. Intermittently vortexing every 2-3 minutes will do a lot of help to accelerate the solubilization.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Note: Buffer DE-A is red liquid, so you can observe the color to make sure the gel is fully dissolved.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Add 0.5× Buffer DE-A volume of Buffer DE-B and mix. If the DNA fragment is less than 400bp, supplement further with a 1×sample volume of isopropanol.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Note: After the addition of DE-B, the solution should be in the uniform color of yellow.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6. Place a Miniprep column into a 2ml microfuge tube (provided). Transfer the solubilized agarose from the step above into the column. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7. Return the Miniprep column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">8. Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">9 Place the Miniprep column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">10. Place the Miniprep column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">11. Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 50ul of ddH2O to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Note</span></strong><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">: Pre-warm the ddH2O at 65</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> will generally improve elution efficiency.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Dot-blot</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1. Spot 1-2 microliter of antigen on to a piece of membrane(</span><span style=";font-family:'Times New Roman';line-height:150%;color:rgb(46,48,51);font-size:14px;background:rgb(249,251,252)">Nitrocellulose nc membrane</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">), let air dry for 30 min or longer;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. Incubate with Blocking buffer for 8-12 hr;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Wash with wash buffer, 3x10min;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. Incubate with primary antibody,1 hr;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Wash with wash buffer, 3x10min;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6. Incubate with enzyme-labeled secondary antibody, 30 min;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7. Wash with wash buffer, 3x10 min;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">8. Add enzyme substrate, wait 5 min;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">9. Detect by eye or with colorimetric or chemiluminescent imaging system.</span> |
| + | </p> |
| + | <table style="width: 557px"> |
| + | <tbody> |
| + | <tr style="height:72px"> |
| + | <td width="177" valign="top" style="width: 177px;padding: 0 7px;border-width: 1px;border-color: windowtext"> |
| + | <p style="text-align:center"> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">Blocking Buffer</span> |
| + | </p> |
| + | </td> |
| + | <td width="198" valign="top" style="width: 198px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top-width: 1px;border-top-color: windowtext;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="text-align:center"> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">Wash Buffer(1L)</span> |
| + | </p> |
| + | </td> |
| + | <td width="181" valign="top" style="width: 182px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top-width: 1px;border-top-color: windowtext;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p style="text-align:center"> |
| + | <span style=";font-family:'Times New Roman';color:rgb(46,48,51);font-size:14px;background:rgb(249,251,252)">Antibody Dilution Buffer</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr style="height:135px"> |
| + | <td width="177" valign="top" style="width: 177px;padding: 0 7px;border-left-width: 1px;border-left-color: windowtext;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">5% Skim milk powder</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">0.5% BSA</span> |
| + | </p> |
| + | </td> |
| + | <td width="198" valign="top" style="width: 198px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p> |
| + | <span style="font-family: 'Times New Roman';font-size: 14px">8g NaCl</span> |
| + | </p> |
| + | <p> |
| + | <span style="font-family: 'Times New Roman';font-size: 14px">0.2g KCl</span> |
| + | </p> |
| + | <p> |
| + | <span style="font-family: 'Times New Roman';font-size: 14px">3g Tris-base</span> |
| + | </p> |
| + | <p> |
| + | <span style="font-family: 'Times New Roman';font-size: 14px">1ml Tween-20</span> |
| + | </p> |
| + | <p> |
| + | <span style="font-family: 'Times New Roman';font-size: 14px">PH=7.4</span> |
| + | </p> |
| + | </td> |
| + | <td width="181" valign="top" style="width: 182px;padding: 0 7px;border-left: none;border-right-width: 1px;border-right-color: windowtext;border-top: none;border-bottom-width: 1px;border-bottom-color: windowtext"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">2.5% Skim milk powder</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">0.5% BSA</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p style="margin-bottom:0;text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">E. Coli calcium chloride competent cells</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1.Inoculate a single colony into 5ml LB in a 50ml Falcon tube. Grow overnight at 37°C.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2.Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3.Shake at 37°C for 1.5-3hrs. OR Inoculate a single colony into 25ml LB in a 250 ml bottle in the morning. and then shake at 37°C for 4-6 hrs.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4.Put the cells on ice for 10 mins (keep cold form now on).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5.Collect the cells by centrifugation for 3 mins at 6000 rpm.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6.Decant the supernatant and gently resuspend on 10 ml cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7.Incubate on ice for 20 mins</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">8.Centrifuge for 3 minutes at 6000 rpm.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">9.Discard supernatant and gently resuspend on 5ml cold 0.1 M CaCl2/15% Glycerol</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">10.Dispense in microtubes (300μl/tube). Freeze in - 80°C.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">CaCI2/15% Glycerol-solutions for competent cells Material</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">0.1 M CaCl2</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">15 % glycerol solution</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Fixation of bacterial cells and fluorescence labeling</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1.Cells from overnight cultures (induced if required) were harvested and washed in PBS (0.145 M NaCl; 0.15 M sodium phosphate).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2.Cells were fixed by mixing 250 ul cells with a 750 u1 4 % (w/v)solution of paraformaldehyde in PBS. This mixture was incubated on ice for 20 min. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3.To remove the fixative, cells were washed twice in PBS.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4.Samples of 20 ul were placed on a poly-L-lysine-coated slide and air-dried.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. After washing in PBS, 16 u1 of a 1: 5 (monoclonal) or 1: 25 (polyclonal) dilution of the primary antiserum was placed on top of each sample and left in a moist incubation chamber for 1h. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6.The slides were washed three times in PBS and 16 ul FITC-conjugated antiserum was added. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7.After 2 h in the dark, the slides were washed three times in PBS, and a drop of Citifluor was placed on top of each sample before microscopy.</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p> |
| + | <strong><span style="font-family: 'Times New Roman';font-size: 14px"> </span></strong> |
| + | </p> |
| + | <p> |
| + | <strong><span style="font-family: 'Times New Roman';font-size: 14px"> </span></strong> |
| + | </p> |
| + | <p> |
| + | <strong><span style="font-family: 'Times New Roman';font-size: 14px"> </span></strong> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">General Heat-Shock Transformation</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1. Add 10ul DNA to 50ul cells on ice (set positive control by using Pcotc, cotc, PtasA, GolB,PbrR DNA fragment and ddH</span><sub><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;vertical-align:sub">2</span></sub><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">O, set negative control by using chemically competent </span><em><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">E.coli</span></em><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> cells without plasmids).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. Incubate on ice for 30 minutes.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Heat shock at 42</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> for exactly 90 seconds.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. Place samples back on ice for 1-2 minutes.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Operating in the clean bench, add 900ul of LB broth per tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6. Incubate at 37</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> for 60 minutes, shaking.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7. Activate it on the plate for 60 minutes. The total number of plates is 7.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">8. Centrifuge at 3000rpm for 1 minute.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">9. Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">10. Use the inoculating loop to load bacteria liquid then streak on the LB plate.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">11. Place plates upside down and incubate at 37</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> overnight.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">LB medium</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1.Dissolve 10 g peptone, 5 g yeast extract, and 5 g NaCl in 950 mL deionized water.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">2.Adjust the pH of the medium to 7.0 using 1M NaOH and bring volume up to 1 liter.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">3.Autoclave on liquid cycle for 20 min at 15 psi. Allow solution to cool to 55°C, and add antibiotic if needed (34µg/ ml of Amp or Kan).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">4.Store at room temperature or +4°C (2).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Material</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">10 g peptone</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">5 g yeast extract</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">5 g NaCl</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">1 M NaOH</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Antibiotic if needed</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">dH2O</span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">LB agar-plates</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1.Prepare LB medium as above, but add 10 g/L agar before autoclaving.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2.After autoclaving, cool to approx. 55°C, add antibiotic (if needed, the concentration of antibiotic to LB should be 1:1000), and pour into petridishes.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3.Let it cool, then invert and store at +4°C in the dark (2).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Material</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">LB medium</span> |
| + | </p> |
| + | <p style="text-align:justify;text-justify:inter-ideograph;line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">10 g/LAgar</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="text-align:center"> |
| + | <strong><span style="font-family: 'Times New Roman';font-size: 14px">PCR method/Taq PCR</span></strong><strong></strong> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">1. Thaw Taq, dNTP, primers, template DNA on ice.</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">2. To a new PCR tube, add:</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <table style="width: 274px;margin-left: 7px"> |
| + | <tbody> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-width: 1px;border-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">template DNA </span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top-width: 1px;border-top-color: rgb(204, 204, 204);border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">0.5ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">dNTP</span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">2ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">10×buffer</span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">2.5ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">Mg</span><sup><span style=";font-family:'Times New Roman';font-size:14px;vertical-align:super">2+</span></sup> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">2ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">F primer</span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">1ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">P primer</span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">1ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">rTaq E</span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">0.5ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">ddH</span><sub><span style="font-family:'Times New Roman';font-size:14px;vertical-align:sub">2</span></sub><span style="font-family:'Times New Roman';font-size:14px">O</span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">15.5ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td width="141" valign="top" style="width: 142px;padding: 10px;border-left-width: 1px;border-left-color: rgb(204, 204, 204);border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">total</span> |
| + | </p> |
| + | </td> |
| + | <td width="132" valign="top" style="width: 132px;padding: 10px;border-left: none;border-right-width: 1px;border-right-color: rgb(204, 204, 204);border-top: none;border-bottom-width: 1px;border-bottom-color: rgb(204, 204, 204)"> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px">25ul</span> |
| + | </p> |
| + | </td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Mix solution well.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. Place tube in PCR thermocycler. Set thermocycler program:</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Inititial denaturation: 3min at 95</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Loop (29 cycles), </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Denaturation: 30s at 95</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">,</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Annealing: 30s at 60</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">, </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Elongation: 1min at 72</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">;</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Final elongation: 10min at 72</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">; </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Store: 12</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">.(not for too long).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. We use 5ul of the PCR product for electrophoresis and 45ul for purification (details see DNA purification/AxyPrep PCR DNA purification PCR).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">Plasmid extraction</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1. Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. Add 250ul of Buffer S1 to the pellet to resuspend bacteria cells.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Add 250ul of Buffer S2, mix gently by inverting the tube 4-6 times until the solution becomes clear. The time should be no longer than 5 minutes. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. Add 350ul of Buffer S3, mix gently by inverting the tube 6-8 times. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Centrifuge at 12,000rpm for 10 minutes.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6. Place spin column into a 2ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7. Return the column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">8. Return the column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">9. Place the column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">10. Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">11. Transfer the column into a clean 1.5ml microfuge tube (provided). Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute. Note: Pre-warm the Eluent or deionized water at 65</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> will generally improve elution efficiency. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)">Preparation of single cell suspension of spleen tissue</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(249,251,252)">Cut up method<span style="font-family:微软雅黑">:</span></span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(238,240,242)">1.Add a small amount of PBS solution and 20% fetal bovine serum after putting the tissue into a plate.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px;background:rgb(238,240,242)">2.The tissue was cut to homogenate by eye scissors, and 5mL of PBS fluid and 20% fetal bovine serum were added.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3.Use straws to absorb tissue homogenized and filter into test tube with 100 mesh stainless steel strainer (other purchase).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4.The centrifuge precipitates </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1, 500 </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">rpm</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> x 3 </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">min,and then cleaned 3 times with the cell cleaning fluid.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Each time, the cell debris is removed by 500 rpm short and low speed centrifuge, and the cell block is filtered by 200 mesh stainless steel strainer (other purchase).</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">6.The cell count and the adjustment of cell concentration were </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">(2 ~ 5) x 107 /</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> mL.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">7.Place at room temperature to measure the activity of cells.The cell concentration was </span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2 x 108~1 x 109 /</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">mL of single-cell suspension with 20% fetal bovine serum or sample diluent before separation.</span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px;background:rgb(249,251,252)"> </span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px;background:rgb(249,251,252)"> </span> |
| + | </p> |
| + | <p> |
| + | <span style=";font-family:'Times New Roman';font-size:14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">protein’s inducing</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1. For the engineered-competent </span><em><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">E.coli</span></em><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> cells stored at -80</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">, culture them on LB-agar medium using streak plate method. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. After culturing on LB-agar at 37</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> for 12 hours, pick a single colony, shake in 3mL LB liquid buffer, 37</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">, 160rpm, for 8 hours over night. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Extract 1mL from 300mL sanitized LB liquid buffer, measure its OD600 as blank. Add the 3mL product of step ii into ~300mL LB liquid buffer. </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. Shake for about 2 hours, 37</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">, 160rpm, until OD600 reaches ~0.6 (OD600 should be measured after 1.5 hours and could be predicted using the formula OD600t+20min=2*OD600t). </span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Add 120uL 1M IPTG.</span><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px;background: rgb(249, 251, 252)"> to a final concentration:1-4 mM.</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> After culturing at 37</span><span style=";font-family:宋体;line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> for 12 hours.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px"> </span> |
| + | </p> |
| + | <p style="text-align:center;line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">SDS-PAGE Electrophoresis</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">According to the size of the target protein, select the appropriate concentration of PAGE separation gel. Table 1 list gel concentration formula.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">I Preparation of separation gel</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1. Different volumes of pure water, 30% Acrylamide, 1.5M Tris-HCI Buffer (PH=8.8)and 10%SDS was added to the centrifuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. Add 10% APS and TEMED, and mix immediately for 5-10 seconds to allow the solution to mix well.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. In the gel mold, quickly poured into the appropriate amount of separation glue solution (for 1 mm mini-gel, separation gel solution plus about 4 ml)</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">And then gently cover a layer of 1-3 cm the water layer, so that the gel surface remained flat.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. A clear interface between the gel and the water indicates that the gel has polymerized.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">II Preparation of concentrated gel</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">Remove the water layer covering the interlayer gel and drain the remaining water with filter paper.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">1. Different volumes of pure water, 30% Acrylamide, 1.5M Tris-HCI Buffer (PH=8.8)and 10%SDS was added to the centrifuge tube.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">2. Add 10% APS and TEMED and mix immediately for 5-10 seconds to allow the solution to mix well.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">3. Insert the comb into the gel to avoid air bubbles.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">4. After gel polymerization, carefully pull out the comb to avoid damaging the fill holes.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">5. Perform electrophoresis.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <strong><span style="font-family: 'Times New Roman';line-height: 150%;font-size: 14px">IV Electrophoresis</span></strong><strong></strong> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">The electrophoresis tank into the 4</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px">℃</span><span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> or ice bath, the outer tank by adding SDS buffer, the inner tank into the SDS buffer, after adding the sample, 250V electrophoresis 5 min, 110V electrophoresis to bromophenol blue to reach the bottom of the gel. After the stop electrophoresis, carried out Coomas bright blue staining or electric transfer.</span> |
| + | </p> |
| + | <p style="line-height:150%"> |
| + | <span style=";font-family:'Times New Roman';line-height:150%;font-size:14px"> </span> |
| + | </p> |
| + | <p> |
| + | <br/> |
| + | </p> |
| + | </div> |
| + | </article> |
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