Team:NCTU Formosa/protocol

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              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol">Mutation</a></li>
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              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_F">Flow Cytometry</a></li>
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<br><br><br>
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<div class = "titleDesign">test title >></div>
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<br><br>
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<div id="blueBox"><p>Protocols</p></div>
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<div id="Box"><h2>Point Mutation</h2>
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<p>The procedure is as follows : </p>
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<ol type="decimal">
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<li> Design primers </li>
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<li> Find the best PCR condition by gradient PCR<br>
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<li> KOD PCR condition<br>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<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>
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KOD enzyme    0.5<br>H2O 17<br>Total 25<br></td>
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<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>
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</tr></table>
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</li>
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<li> Confirm the PCR product with electrophoresis</li>
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<li> DPN1  37℃ for 3hr~overnight <br>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<tr><td> DPN1 0.5<br>Buffer2 2<br>PCR product 17.5<br>Total 20<br>
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</td></tr></table>
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</li>
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<li> 80℃ 20mins to denature the DPN1</li>
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<li> Confirm with electrophoresis </li>
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<li> Self ligation room temperature for 2~3 hr <br>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<tr><td> Enzyme 1<br> Buffer 2<br> ATP 2<br> H2O 5<br> PCR product    10<br>Total 20<br>
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</td></tr></table>
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</li>
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<li> Transform DH5alpha with the self-ligation product <br>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<tr><td> Self-ligation product  20<br>DH5alfa 50<br>
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</td></tr></table>
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</li>
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<li> Incubate in Ap25 plate then transfer to Ap50 plate</li>
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</ol>
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</div>
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Latest revision as of 13:41, 5 October 2011



Protocols

Point Mutation

The procedure is as follows :

  1. Design primers
  2. Find the best PCR condition by gradient PCR
  3. 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 25
    		 94℃ 5 min
    94℃ 30 sec
    55℃ 30 sec
    72℃ 5 min
    72℃ 7~10 min
    Cycles : 25
  4. Confirm the PCR product with electrophoresis
  5. DPN1 37℃ for 3hr~overnight
    DPN1 0.5
    Buffer2 2
    PCR product 17.5
    Total 20
  6. 80℃ 20mins to denature the DPN1
  7. Confirm with electrophoresis
  8. Self ligation room temperature for 2~3 hr
    Enzyme 1
    Buffer 2
    ATP 2
    H2O 5
    PCR product 10
    Total 20
  9. Transform DH5alpha with the self-ligation product
    Self-ligation product 20
    DH5alfa 50
  10. Incubate in Ap25 plate then transfer to Ap50 plate

Retrieved from "http://2011.igem.org/Team:NCTU_Formosa/protocol"