User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/07

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September 7th, 2010

1. Prepare XbaI restriction to BBa_I20260 Δ RBS.

  • Double restriction methods:
DNA -> 15 ul
Buffer 2 -> 4 ul (10% of total volume)
BSA -> 1 ul
XbaI -> 2 ul
HPLC -> 18 ul (to complete total volume of 20ul)
Incubate at 37ºC 3.5 hrs

2. Inactivate restriction enzyme, 20 min at 65ºC.

3. Make ligation of BBa_I20260 with the new RBS.

  • Ligation methods (Total volume 20ul):
DNA -> 7ul
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.