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Team:NCTU Formosa/protocol
From 2011.igem.org
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| + | <div id="banner"><a href = "https://2011.igem.org/Team:NCTU_Formosa"><img src = "https://static.igem.org/mediawiki/2011/8/8c/Banner010.jpg" height="236" width="944"/></a></div> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_data">Data</a></li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_discussion"> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_discussion">Modeling</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_data">Data</a></li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_discussion"> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_discussion">Modeling</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_data">Data</a></li> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_data">Data</a></li> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_data">Data</a></li> | ||
| - | |||
</ul> | </ul> | ||
</li> | </li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/modeling"> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/modeling">Measurements</a></li> |
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/parts">Parts</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/parts">Parts</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/safty">Safety</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/safty">Safety</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/humanpractice">Human Practice</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/humanpractice">Human Practice</a></li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/contributions"> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/contributions">Attribution</a></li> |
<li><a class="arrow no-click">Notebook </a> | <li><a class="arrow no-click">Notebook </a> | ||
<ul> | <ul> | ||
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<ul> | <ul> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol">Mutation</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol">Mutation</a></li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_F">Flow</a></li> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_F">Flow Cytometry</a></li> |
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_G">GC</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_G">GC</a></li> | ||
</ul> | </ul> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
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<br><br> | <br><br> | ||
| + | <div id="blueBox"><p>Protocols</p></div> | ||
| + | <div id="Box"><h2>Mutation</h2> | ||
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| + | <p>The procedure is as follows : </p> | ||
| + | <ol type="decimal"> | ||
| + | <li> Design primers </li> | ||
| + | <li> Find the best PCR condition by gradient PCR<br> | ||
| + | <li> KOD PCR condition<br> | ||
| + | <table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all"> | ||
| + | <tr><td> Plasmid 0.5 μl<br>pF 0.5<br>pR 0.5<br>MgCl2 1<br>dNTP 2.5<br>buffer 2.5<br> | ||
| + | KOD enzyme 0.5<br>H2O 17<br>Total 25<br></td> | ||
| + | <td>94℃ 5 min<br>94℃ 30 sec<br>55℃ 30 sec<br>72℃ 5 min<br>72℃ 7~10 min<br>Cycles : 25<br><br><br><br></td> | ||
| + | </tr></table> | ||
| + | </li> | ||
| + | <li> Confirm the PCR product with electrophoresis</li> | ||
| + | <li> DPN1 37℃ for 3hr~overnight <br> | ||
| + | <table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all"> | ||
| + | <tr><td> DPN1 0.5<br>Buffer2 2<br>PCR product 17.5<br>Total 20<br> | ||
| + | </td></tr></table> | ||
| + | </li> | ||
| + | <li> 80℃ 20mins to denature the DPN1</li> | ||
| + | <li> Confirm with electrophoresis </li> | ||
| + | <li> Self ligation room temperature for 2~3 hr <br> | ||
| + | <table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all"> | ||
| + | <tr><td> Enzyme 1<br> Buffer 2<br> ATP 2<br> H2O 5<br> PCR product 10<br>Total 20<br> | ||
| + | </td></tr></table> | ||
| + | </li> | ||
| + | <li> Transform DH5alpha with the self-ligation product <br> | ||
| + | <table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all"> | ||
| + | <tr><td> Self-ligation product 20<br>DH5alfa 50<br> | ||
| + | </td></tr></table> | ||
| + | </li> | ||
| + | <li> Incubate in Ap25 plate then transfer to Ap50 plate</li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | </body> | ||
| + | </html> | ||
Revision as of 20:02, 2 October 2011
Protocols
Mutation
The procedure is as follows :
- Design primers
- Find the best PCR condition by gradient PCR
- KOD PCR condition
Plasmid 0.5 μl
pF 0.5
pR 0.5
MgCl2 1
dNTP 2.5
buffer 2.5
KOD enzyme 0.5
H2O 17
Total 2594℃ 5 min
94℃ 30 sec
55℃ 30 sec
72℃ 5 min
72℃ 7~10 min
Cycles : 25 - Confirm the PCR product with electrophoresis
- DPN1 37℃ for 3hr~overnight
DPN1 0.5
Buffer2 2
PCR product 17.5
Total 20
- 80℃ 20mins to denature the DPN1
- Confirm with electrophoresis
- Self ligation room temperature for 2~3 hr
Enzyme 1
Buffer 2
ATP 2
H2O 5
PCR product 10
Total 20
- Transform DH5alpha with the self-ligation product
Self-ligation product 20
DH5alfa 50
- Incubate in Ap25 plate then transfer to Ap50 plate
