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Team:UESTC-China/Protocol
From 2014.igem.org
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| - | #SensorEditingArea{position:absolute; left:0px; top:300px; height: | + | #SensorEditingArea{position:absolute; left:0px; top:300px; height:6500px; width:1183px; background-color:#ffffff; padding:60px 0px 0px 30px;} |
.SensorEditingAreaClass p{position:relative;left:0px; width:1140px; font-size:23px; font-family: calibri, arial, helvetica, sans-serif; text-align:justify;} | .SensorEditingAreaClass p{position:relative;left:0px; width:1140px; font-size:23px; font-family: calibri, arial, helvetica, sans-serif; text-align:justify;} | ||
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#SectionTitles1{ | #SectionTitles1{ | ||
position:relative; | position:relative; | ||
| - | left: | + | left:0px; |
background-color:#1f8a70; | background-color:#1f8a70; | ||
padding:0 10px; | padding:0 10px; | ||
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font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | ||
#SectionTitles7{ | #SectionTitles7{ | ||
| - | top: | + | top: 2400px; |
position:relative; | position:relative; | ||
left:30px; | left:30px; | ||
| + | background-color:#1f8a70; | ||
| + | padding:0 10px; | ||
| + | height:40px; | ||
| + | line-height:40px; | ||
| + | font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | ||
| + | #SectionTitles8{ | ||
| + | top: 2800px; | ||
| + | position:relative; | ||
| + | left:30px; | ||
| + | background-color:#1f8a70; | ||
padding:0 10px; | padding:0 10px; | ||
height:40px; | height:40px; | ||
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font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | ||
| + | #SectionTitles9{ | ||
| + | top: 3200px; | ||
| + | position:relative; | ||
| + | left:30px; | ||
| + | padding:0 10px; | ||
| + | height:40px; | ||
| + | line-height:40px; | ||
| + | font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | ||
#BiosensorsTable{position:absolute;margin-top: 20px; left:0px; width:1000px; font-family:calibri;arial;helvetica;sans-serif; font-size:18px; text-align:center; } | #BiosensorsTable{position:absolute;margin-top: 20px; left:0px; width:1000px; font-family:calibri;arial;helvetica;sans-serif; font-size:18px; text-align:center; } | ||
.mid{background: #FFFFCC;color: #797268; } | .mid{background: #FFFFCC;color: #797268; } | ||
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<body> | <body> | ||
<div id="total-wrap"> | <div id="total-wrap"> | ||
| - | + | <div id="header"> | |
| - | + | <div id="emp"> | |
| - | + | <div id='logo'><a href="https://igem.org"></a></div> | |
| + | </div> | ||
| + | <div id="top-menu"> | ||
| + | <div class="top-menu-each"> <a href="https://2014.igem.org/Team:UESTC-China"> | ||
| + | <p>Home</p> | ||
| + | </a> </div> | ||
| + | <div class="top-menu-each"> | ||
| + | <div class="arrow"></div> | ||
| + | <p>Team</p> | ||
| + | <div class="top-menu-sub"> | ||
| + | <ul> | ||
| + | <a href="https://2014.igem.org/Team:UESTC-China/Team"> | ||
| + | <li>Members</li> | ||
| + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Attribution"> | ||
| + | <li>Attribution</li> | ||
| + | </a> <a href="https://igem.org/Team.cgi?year=2014&team_name=UESTC-China"> | ||
| + | <li>Team Profile</li> | ||
| + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Notebook"> | ||
| + | <li>Notebook</li> | ||
| + | </a> | ||
| + | </ul> | ||
| + | </div> | ||
</div> | </div> | ||
| - | + | <div class="top-menu-each"> | |
| - | + | <div class="arrow"></div> | |
| - | + | <p>Project</p> | |
| - | + | <div class="top-menu-sub"> | |
| - | + | <ul> | |
| - | + | <a href="https://2014.igem.org/Team:UESTC-China/Project"> | |
| - | + | <li>Overview</li> | |
| - | + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Design"> | |
| - | + | <li>Design</li> | |
| - | + | </a> <a href="https://2014.igem.org/Team:UESTC-China/result"> | |
| - | + | <li>Result</li> | |
| - | + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Futurework"> | |
| - | + | <li>Future work</li> | |
| - | + | </a> | |
| - | + | </ul> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
</div> | </div> | ||
| - | + | </div> | |
| - | + | <div class="top-menu-each"> | |
| - | + | <div class="arrow"></div> | |
| - | + | <p>Modeling</p> | |
| - | + | <div class="top-menu-sub"> | |
| - | + | <ul> | |
| - | + | <a href="https://2014.igem.org/Team:UESTC-China/Modeling"> | |
| - | + | <li>Modeling</li> | |
| - | + | </a> | |
| - | + | </ul> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
</div> | </div> | ||
| - | + | </div> | |
| - | + | <div class="top-menu-each"> | |
| - | + | <div class="arrow"></div> | |
| - | + | <p>Parts</p> | |
| - | + | <div class="top-menu-sub"> | |
| - | + | <ul> | |
| - | + | <a href="https://2014.igem.org/Team:UESTC-China/Material"> | |
| - | + | <li>BioBrick</li> | |
| - | </ | + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Protocol"> |
| - | </ | + | <li>Protocol</li> |
| + | </a> | ||
| + | </ul> | ||
</div> | </div> | ||
| - | + | </div> | |
| - | + | <div class="top-menu-each"> | |
| - | + | <div class="arrow"></div> | |
| - | + | <p>Human Practice</p> | |
| - | + | <div class="top-menu-sub"> | |
| - | + | <ul> | |
| - | + | <a href="https://2014.igem.org/Team:UESTC-China/HumanPractice.html"> | |
| - | + | <li>Knowledge quiz</li> | |
| - | + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Lecture.html"> | |
| - | + | <li>Lecture</li> | |
| - | </ | + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Communication.html"> |
| - | </ | + | <li>Communication</li> |
| + | </a> <a href="https://2014.igem.org/Team:UESTC-China/Questionnaire.html"> | ||
| + | <li>Questionnaire</li> | ||
| + | </a> | ||
| + | </ul> | ||
</div> | </div> | ||
| - | + | </div> | |
| - | + | <div class="top-menu-each"> | |
| - | + | <div class="arrow"></div> | |
| - | + | <p>Safety</p> | |
| - | + | <div class="top-menu-sub"> | |
| - | + | <ul> | |
| - | + | <a href="https://2014.igem.org/Team:UESTC-China/Safety"> | |
| - | + | <li>Safety</li> | |
| - | + | </a> | |
| - | + | </ul> | |
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</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
| + | <div id="middle-photo"> | ||
| + | <div id="SensorEditingArea" class="SensorEditingAreaClass"> | ||
| + | <div id="SectionTitles1" style="width:500px;">DNA ligation with T4 DNA Ligase | ||
| + | </h1> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top">Component</th> | ||
| + | <th class="top">20ul Reaction</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">1.33X Master Mix</td> | ||
| + | <td class="mid"> 15ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Insert DNA </td> | ||
| + | <td class="mid"> the moles of insert DNA to vector DNA is 5：1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">>Vector DNA</td> | ||
| + | <td class="mid"> 50ng</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid"> To 20ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Incubate at 50℃ for 1 hour</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles2" style="width:500px;">DNA ligation with Gibson Assemble | ||
| + | </h1> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top">Component</th> | ||
| + | <th class="top">20ul Reaction </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Vector DNA</td> | ||
| + | <td class="mid">50ng</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Insert DNA</td> | ||
| + | <td class="mid"> the moles of insert DNA to vector DNA is 5：1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10X T4 DNA ligase buffer</td> | ||
| + | <td class="mid">2ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">T4 DNA ligase</td> | ||
| + | <td class="mid">1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid">to 20ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Incubate at 25℃ for 12 hours</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles3" style="width:500px;">Double Digestion of DNA | ||
| + | </h1> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top">Component</th> | ||
| + | <th class="top">50ul Reaction </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10x FastDigest buffer</td> | ||
| + | <td class="mid">5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Restriction enzyme 1</td> | ||
| + | <td class="mid"> 1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Restriction enzyme 2</td> | ||
| + | <td class="mid">1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">DNA</td> | ||
| + | <td class="mid">1-2ug</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid">to 50ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Incubate at 37℃ for 2 hours</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles4" style="width:500px;">PCR protocol for KOD-Plus-Neo | ||
| + | </h1> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top">Component</th> | ||
| + | <th class="top">50ul Reaction </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid">33ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10X buffer for KOD-Plus-Neo</td> | ||
| + | <td class="mid"> 5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">MgSO4</td> | ||
| + | <td class="mid">1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10mM dNTPs</td> | ||
| + | <td class="mid">1ug</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM forward primer</td> | ||
| + | <td class="mid">1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM reverse primer</td> | ||
| + | <td class="mid"> 1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Template</td> | ||
| + | <td class="mid">1ul </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">KOD enzyme</td> | ||
| + | <td class="mid"> 1ul</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles5" style="width:500px;"> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top"></th> | ||
| + | <th class="top">Temperature </th> | ||
| + | <th class="top">Time </th> | ||
| + | <th class="top"> Cycle</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step1</td> | ||
| + | <td class="mid">94℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid">X35 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid"> 56℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">68℃</td> | ||
| + | <td class="mid">30s/kb</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step3</td> | ||
| + | <td class="mid">68℃</td> | ||
| + | <td class="mid">5min</td> | ||
| + | <td class="mid">X1 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">10℃</td> | ||
| + | <td class="mid">10min</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM reverse primer</td> | ||
| + | <td class="mid"> 1ul</td> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid">33ul</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles6" style="width:500px;">PCR protocol for KOD-Plus-Neo | ||
| + | </h1> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top">Component</th> | ||
| + | <th class="top">50ul Reaction </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid">32ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10X buffer for KOD-Plus-Neo</td> | ||
| + | <td class="mid"> 5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">MgSO4</td> | ||
| + | <td class="mid">3ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10mM dNTPs</td> | ||
| + | <td class="mid">5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM forward primer</td> | ||
| + | <td class="mid">1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM reverse primer</td> | ||
| + | <td class="mid"> 1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Template</td> | ||
| + | <td class="mid">1ul </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">KOD enzyme</td> | ||
| + | <td class="mid"> 1ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Each template will be diluted by the number of moles into 2-10ng/ul.</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles7" style="width:500px;"> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top"></th> | ||
| + | <th class="top">Temperature </th> | ||
| + | <th class="top">Time </th> | ||
| + | <th class="top"> Cycle</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step1</td> | ||
| + | <td class="mid">94℃</td> | ||
| + | <td class="mid">5min</td> | ||
| + | <td class="mid">X1 cycle</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step2</td> | ||
| + | <td class="mid"> 94℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid">X35 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">56℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">68℃</td> | ||
| + | <td class="mid">30s/kb</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step3</td> | ||
| + | <td class="mid"> 68℃</td> | ||
| + | <td class="mid">5min</td> | ||
| + | <td class="mid">X1 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid"> 10℃</td> | ||
| + | <td class="mid">10min</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles8" style="width:500px;">Colony PCR with Taq DNA polymerase | ||
| + | </h1> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top">Component</th> | ||
| + | <th class="top">25ul Reaction </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid">15.8ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10X Taq buffer</td> | ||
| + | <td class="mid"> 2.5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">2.mM dNTPs</td> | ||
| + | <td class="mid">0.5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM forward primer</td> | ||
| + | <td class="mid">0.5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM reverse primer</td> | ||
| + | <td class="mid">0.5ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Taq enzyme</td> | ||
| + | <td class="mid"> 0.2ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Water with colony </td> | ||
| + | <td class="mid">5ul </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles9" style="width:500px;"> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top"></th> | ||
| + | <th class="top">Temperature </th> | ||
| + | <th class="top">Time </th> | ||
| + | <th class="top"> Cycle</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step1</td> | ||
| + | <td class="mid">95℃</td> | ||
| + | <td class="mid">5min</td> | ||
| + | <td class="mid">X1 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step2</td> | ||
| + | <td class="mid"> 94℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid">X35 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">56℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">72℃</td> | ||
| + | <td class="mid">1min/kb</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step3</td> | ||
| + | <td class="mid">72℃</td> | ||
| + | <td class="mid">10min</td> | ||
| + | <td class="mid">X1 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">10℃</td> | ||
| + | <td class="mid">10min</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </div> | ||
</div> | </div> | ||
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Revision as of 14:59, 24 September 2014
DNA ligation with T4 DNA Ligase
| Component | 20ul Reaction |
|---|---|
| 1.33X Master Mix | 15ul |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| >Vector DNA | 50ng |
| ddH2O | To 20ul |
| Incubate at 50℃ for 1 hour |
DNA ligation with Gibson Assemble
| Component | 20ul Reaction |
|---|---|
| Vector DNA | 50ng |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| 10X T4 DNA ligase buffer | 2ul |
| T4 DNA ligase | 1ul |
| ddH2O | to 20ul |
| Incubate at 25℃ for 12 hours |
Double Digestion of DNA
| Component | 50ul Reaction |
|---|---|
| 10x FastDigest buffer | 5ul |
| Restriction enzyme 1 | 1ul |
| Restriction enzyme 2 | 1ul |
| DNA | 1-2ug |
| ddH2O | to 50ul |
| Incubate at 37℃ for 2 hours |
PCR protocol for KOD-Plus-Neo
| Component | 50ul Reaction |
|---|---|
| ddH2O | 33ul |
| 10X buffer for KOD-Plus-Neo | 5ul |
| MgSO4 | 1ul |
| 10mM dNTPs | 1ug |
| 10uM forward primer | 1ul |
| 10uM reverse primer | 1ul |
| Template | 1ul |
| KOD enzyme | 1ul |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 68℃ | 30s/kb | ||
| Step3 | 68℃ | 5min | X1 cycles |
| 10℃ | 10min | ||
| 10uM reverse primer | 1ul | ddH2O | 33ul |
PCR protocol for KOD-Plus-Neo
| Component | 50ul Reaction |
|---|---|
| ddH2O | 32ul |
| 10X buffer for KOD-Plus-Neo | 5ul |
| MgSO4 | 3ul |
| 10mM dNTPs | 5ul |
| 10uM forward primer | 1ul |
| 10uM reverse primer | 1ul |
| Template | 1ul |
| KOD enzyme | 1ul |
| Each template will be diluted by the number of moles into 2-10ng/ul. |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 94℃ | 5min | X1 cycle |
| Step2 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 68℃ | 30s/kb | ||
| Step3 | 68℃ | 5min | X1 cycles |
| 10℃ | 10min |
Colony PCR with Taq DNA polymerase
| Component | 25ul Reaction |
|---|---|
| ddH2O | 15.8ul |
| 10X Taq buffer | 2.5ul |
| 2.mM dNTPs | 0.5ul |
| 10uM forward primer | 0.5ul |
| 10uM reverse primer | 0.5ul |
| Taq enzyme | 0.2ul |
| Water with colony | 5ul |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 95℃ | 5min | X1 cycles |
| Step2 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 72℃ | 1min/kb | ||
| Step3 | 72℃ | 10min | X1 cycles |
| 10℃ | 10min |
