User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/11

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October 11th, 2010

1. Inactivate restriction enzyme for 20 min at 80º C.

2. Make the following ligations:

  • pSB3K3 + J23101 SpeI-PstI (3ul) with ΔRBS + GFP E004 XbaI-PstI (5ul)
  • pSB4A5 + MinBP SpeI-PstI (3ul) with ΔRBS + GFP E004 XbaI-PstI (5ul)
  • 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.