User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/22

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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.