Team:UNIPV-Pavia/Methods Materials/Ligation

From 2009.igem.org

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*'''Roche T4 Ligase'''
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*'''10X Roche T4 Ligase Buffer'''
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'''Roche T4 Ligase'''<hr color='white' width='70%'>
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*'''ddH2O'''
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'''10X Roche T4 Ligase Buffer'''<hr color='white' width='70%'>
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'''ddH2O'''</font>
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Revision as of 16:37, 22 September 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.






Roche T4 Ligase
10X Roche T4 Ligase Buffer

ddH2O





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