User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/27

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September 27th, 2010

1. Inactivate restriction enzyme at 80ºC for 20 min.

2.Make the following ligations:

  • BBa_j61002 plasmid containing promoter J23101 (SpeI-PstI restriction) (3 ul) + Δ RBS-GFP E0040 (5 ul)
  • Religation of pSB3K3 + J23101 (SpeI restriction) (4 ul).
  • Ligation methods (Total volume 20ul):
DNA -> Volume of each part to be ligated is indicated above.
Buffer for T4 DNA ligase 5X -> 4ul (Final concentration 20%)
T4 DNA ligase -> 1ul
HPLC -> Complete total volume (20ul)
Incubate overnight at 16ºC
Mix well (vortex) buffer and reaction tubes, unfreeze buffer on ice.