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 the leu2D1 or wt LEU2 allele present at the genomic LEU2 locus? The second set of PCR primers is designed to identify whether the Sikowski vector is at the genomic locus.

1. Typically, 3 transformants are screened per desired strain. Re-streak each colony onto the appropriate selection plate.
2. Pick a colony at least 2 mm in diameter using a wooden toothpick. Resuspend it into 11 uL Lyse-N-Go (Pierce) in a thick-walled PCR tube. You will need an additional empty PCR tube for each colony.
3. Load all the tubes into the PCR machine and run the Lyse N Go PCR protocol.

• • 65 C for 0.5 min
  • 8 C for 0.5 min
  • 65 C for 1.5 min
  • 97 C for 3 min
  • 8 C for 1 min
  • 65 C for 3 min
  • 97 C for 1 min
  • 65 C for 1 min
  • 80 C forever

1. Immediately after the final cycling step, resuspend the lysate with a pipet, and transfer 5 uL of it to the second PCR tube. Do this while both tubes are in the PCR block at 80 C.
2. Add 45 uL PCR mix. This is composed of:

• • 0.125 uL 100 uM forward primer (see table below)
  • 0.125 uL 100 uM reverse primer (see table below)
  • 1 uL 10 mM dNTP mix (each dNTP is 10 mM)
  • 0.5 uL Taq DNA polymerase (Fischer Scientific)
  • 5 uL 10x Buffer A (Fischer Scientific)
  • 38.25 uL ddH2O

1. Run the following PCR cycle:

• • 95 C for 2 min
  • 95 C for 0.5 min
  • 50 C for 0.5 min
  • 72 C for 2 min
  • 72 C for 9 min
  • 8 C forever

1. Run the PCR products on an agarose gel. Since the bands are typically rather faint, pour the gel as thick as possible, and load 35 uL PCR + dye in the 5 mm-wide lanes.

• Sikorski vector	insert & vector digest
  pRS304* (TRP1)	EcoRI, SpeI
  pRS305 (LEU2)	XbaI, PstI
  pRS306 (URA3)	EcoRI, SpeI

  • 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.