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.
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