User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/23

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August 23rd, 2010

1. Run gel to verify parts to be ligated.

Pre-ligation 23Aug2010.JPG

Lanes: 1) Green ladder; 2) p30-MinBP PstI-SpeI; 3) pSB3K3 EcoRI-PstI; 4) Blue Promoter (PCR product); 5) GFP BBa_K145015 (74 min) XbaI-PstI retsriction.

  • I will only do L2: p30-MinBP + GFP BBa_K145015 (74 min), because the PCR product for the Blue Promoter is not well defined.

2. Make ligation 2: p30-MinBP + GFP BBa_K145015 (74 min).

  • Ligation methods (Total volume 20ul):
DNA -> 5ul of p30-MinBP; 7ul GFP BBa_K145015 (74 min)
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.