Team:UESTC-China/Protocol

From 2014.igem.org

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         </table>
         </table>
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</div>     </div>
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</div>    
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<div id="SectionTitles6" style="width:500px;">PCR protocol for KOD-Plus-Neo
 +
        </h1>
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        <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>
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</tr>
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        </table>
 +
</div>
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<div id="SectionTitles7" style="width:500px;">
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        <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>
</div>
</div>
</div>
</div>

Revision as of 06:07, 24 September 2014

UESTC-China

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

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