Harvard BioDesign/12 June 2014

From 2014.igem.org

PCR

Primers from John→ HBD 1-4

HBD 1 & 2 = fwd and rev for pBbE1a-CsgA backbone HBD 3 & 4 = fwd and rev for Tet promoter

Backbone template from Bom → pBbE1a-CsgA @ 65ng/uL For thet Tet promoter template, we did a colony PCR since we do not have a miniprepped sample. We got this colony from Peter’s plate Bba_I13522 pSB1a2 which has the Tet promoter.

We ran the PCR products on a gel, excised the bands for backbone and promoter for gel purification for Gibson fragments:

After setting up and running the Gibson, we transformed the reaction into MACH1 cells by electroporation and into Turbo cells by heat shock. Plated all transformations on LB+Carb plates @ 5uL and 50uL.

We picked pBbE1a CsgA colonies from Bom’s MACH1 plate and grew them up in 5mL LB+Carb for miniprep since the cultures we inoculated from arc’ed cells did not grow up for minipreps