Team:UNIPV-Pavia/Methods Materials/Ligation

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Revision as of 15:20, 19 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.






Roche T4 Ligase
10X Roche T4 Ligase Buffer

ddH2O





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