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Team:NCTU Formosa/protocol
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<ul id="cm-nav"> | <ul id="cm-nav"> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa">Home</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa">Home</a></li> | ||
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<ul> | <ul> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/members">Members</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/members">Members</a></li> | ||
| - | <li><a href=" | + | <li><a href="http://www.nctu.edu.tw/english/index.php">School</a></li> |
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/gallery">Gallery</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/gallery">Gallery</a></li> | ||
</ul> | </ul> | ||
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| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">Project</a> |
<ul class="arrow-pad"> | <ul class="arrow-pad"> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/introduction">Introduction</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/introduction">Introduction</a></li> | ||
| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">RNA Thermometer</a> |
<ul> | <ul> | ||
<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> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">CI promoter </a> |
<ul> | <ul> | ||
<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> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">Carotenoid synthesis pathway</a> |
<ul> | <ul> | ||
<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> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">Butanol pathway</a> |
<ul> | <ul> | ||
<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> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">Violacein pathway</a> |
<ul> | <ul> | ||
<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> | ||
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<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">Attribution</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/contributions">Attribution</a></li> | ||
| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">Notebook </a> |
<ul> | <ul> | ||
| - | <li><a class="arrow | + | <li><a onClick="out('cm-nav')" class="arrow">Protocols</a> |
<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> | ||
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<br><br> | <br><br> | ||
<div id="blueBox"><p>Protocols</p></div> | <div id="blueBox"><p>Protocols</p></div> | ||
| - | <div id="Box"><h2>Mutation</h2> | + | <div id="Box"><h2>Point Mutation</h2> |
Latest revision as of 13:41, 5 October 2011
Protocols
Point 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
