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Team:UESTC-China/Protocol
From 2014.igem.org
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<tr> | <tr> | ||
<td class="mid">Incubate at 25℃ for 12 hours</td> | <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> | <td class="mid"> </td> | ||
Revision as of 04:31, 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 |
