Team:NCTU Formosa/protocol

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         <li><a href="https://2011.igem.org/Team:NCTU_Formosa/introduction">Introduction</a></li>
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             </ul>
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             </ul>
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               <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>
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<div id="Box"><h2>Mutation</h2>
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<div id="Box"><h2>Point Mutation</h2>

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

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