October 22, 2010
1. Measure in nanodrop the DNA concentration of the parts that will be submitted to the regystry of standard biological parts.
- Minimum Blue Light Receptor Promoter BBa_K360041, this part is in pSB4A5 -> 88.9ng/ul
- Minimum Blue Light Receptor Promoter BBa_K360041 + Strong RBS with spacer BBa_K360031 + GFP BBa_E0040, the name of the whole composite part is BBa_K360141 and is in pSB4A5 -> 123.4 ng/ul
2. Replate in solid LB medium with Cm 10 colonies of transformation made on October 21, pSB1C3 + MinBP + ΔRBS + GFP E004.
3. Make the following ligation:
- pSB3K3 + J23101 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 10X -> 2ul (Final concentration 10%)
- T4 DNA ligase -> 1ul
- HPLC -> Complete total volume (20ul)
- Incubate overnight at 16ºC
- Mix well (vortex) buffer and reaction tubes, unfreeze buffer on ice.
4. Incubate in liquid LB medium with CM 10 colonies of the transformation pSB1C3 + MinBP + ΔRBS + GFP E004. This colonies will be used to extract plasmid.