Team:UNIPV-Pavia/Methods Materials/Ligation

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<h1>Ligation</h1>
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<img width="100%" height="180px" src="https://static.igem.org/mediawiki/2009/d/de/UNIPV_title_M%26M.jpg" ></img>
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{{UNIPV-Pavia/Menu}}
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    <td align="center"><br><bR>
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    <font face="Pristina" size="50" ><b>Protocols</b></font>
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  <td> {{UNIPV-Pavia/Protocols}}</td>
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=='''Ligation'''==
''(estimated time: 20 min + 12-16 hours overnight incubation)''
''(estimated time: 20 min + 12-16 hours overnight incubation)''
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'''Materials needed:'''
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*'''Roche T4 Ligase'''
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*'''10X Roche T4 Ligase Buffer'''
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*'''ddH2O'''
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*(For every ligation)
*(For every ligation)
*Add 50 ng of vector
*Add 50 ng of vector
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*Add   <html><img src="https://static.igem.org/mediawiki/2008/8/82/Pv_formula_lig.png"></img></html>
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*Add <html>&nbsp; &nbsp; &nbsp; <img src="https://static.igem.org/mediawiki/2008/8/82/Pv_formula_lig.png"></img></html>
*Heat DNA mix at 65°C for 5 min for DNA denaturation
*Heat DNA mix at 65°C for 5 min for DNA denaturation
*Add 1 µl of T4 Ligase buffer
*Add 1 µl of T4 Ligase buffer
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*Before transformation you have to inactivate T4 Ligase:
*Before transformation you have to inactivate T4 Ligase:
**Heat ligation at 65°C for 10 min.
**Heat ligation at 65°C for 10 min.
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<tr bgcolor='yellow'><td>INGREDIENTS:
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'''Roche T4 Ligase'''<hr color='white' width='70%'>
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'''10X Roche T4 Ligase Buffer'''<hr color='white' width='70%'>
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'''ddH2O'''</font>
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Latest revision as of 10:33, 21 October 2009

EthanolPVanimation.gif



Protocols

Ligation

(estimated time: 20 min + 12-16 hours overnight incubation)

  • (For every ligation)
  • Add 50 ng of vector
  • Add      
  • Heat DNA mix at 65°C for 5 min for DNA denaturation
  • Add 1 µl of T4 Ligase buffer
  • Add 1 µl of T4 Ligase
  • 10 µl final volume
  • Incubate at 16°C overnight


  • Then, ligation can be conserved at 4°C or can be transformed
  • Before transformation you have to inactivate T4 Ligase:
    • Heat ligation at 65°C for 10 min.






INGREDIENTS:

Roche T4 Ligase
10X Roche T4 Ligase Buffer

ddH2O





Retrieved from "http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Ligation"