Silver: Verifying Genomic Integration

Verifying Genomic Integration

• Single colony PCR is used to identify transformants which have the gene of interest integrated at the genomic locus.
• One set of PCR primers is designed to identify whether the prototrophic or auxotrophic allele is at the genomic locus, e.g. Is leu2D1 or wt LEU2 present at the genomic LEU2 locus? The second set of PCR primers is designed to identify whether the Sikowski vector are at the genomic locus, e.g. Are there regions of the Sikowski vector at the LEU2 locus?
• Thaw competent cells (TOP10 from Invitrogen) on ice.

1. Place 10-15 µL cells in pre-chilled Eppendorf tubes.
2. Add 2 µL BioBrick vector (either native or ligated; so that vector is ~10-20% of the final volume), and chill on ice for 30 min.
   • Do not pipet or vortex.
3. Heat shock at 42 °C for 30 s.
4. Incubate on ice for 2 min.
5. Add 170 µL SOC medium (~10 x volume; Invitrogen), and shake at 37 °C for 15 min for Amp selection, or 30 min for Kan selection.
6. Add all media to a selectable marker LB plate. Use glass beads to streak the plate.
7. Incubate at 37 °C. Transformants should appear within 12 hrs.