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Team:UESTC-China/Protocol
From 2014.igem.org
(Difference between revisions)
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| - | top: | + | top: 700px; |
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#SectionTitles4{ | #SectionTitles4{ | ||
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</div> | </div> | ||
</div> | </div> | ||
| - | + | <div id="SectionTitles2" style="width:500px;">DNA ligation with Gibson Assemble | |
</h1> | </h1> | ||
<table border="1" id="BiosensorsTable" cellspacing="0";> | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
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</table> | </table> | ||
</div> | </div> | ||
| - | + | <div id="SectionTitles3" style="width:500px;">Double Digestion of DNA | |
</h1> | </h1> | ||
<table border="1" id="BiosensorsTable" cellspacing="0";> | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
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</tr> | </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> | </table> | ||
</div> | </div> | ||
Revision as of 04:43, 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 |
