Team:UNIPV-Pavia/Protocols/Ligation

From 2008.igem.org

(Difference between revisions)
Line 40: Line 40:
*Heat vector at 65°C for 5 min for DNA denaturation
*Heat vector at 65°C for 5 min for DNA denaturation
*Add 50 µg of vector
*Add 50 µg of vector
-
*Add <math>\6*50*length(insert)\/length(vector)</math>
+
*Add [[Image:pv_formula_lig.png]]
*Add 1 µl of T4 Ligase buffer
*Add 1 µl of T4 Ligase buffer
*Add 1 µl of T4 Ligase
*Add 1 µl of T4 Ligase

Revision as of 12:47, 1 July 2008

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The protocols we used


Ligation

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

Materials needed:

  • Roche T4 Ligase
  • Roche T4 Ligase Buffer
  • ddH2O


  • (For every ligation)
  • Heat vector at 65°C for 5 min for DNA denaturation
  • Add 50 µg of vector
  • Add Pv formula lig.png
  • Add 1 µl of T4 Ligase buffer
  • Add 1 µl of T4 Ligase
  • 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.