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.
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