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. In this case, one primer binds to the genomic DNA and the second primer binds to the coding sequence of the auxotrophic gene. The second set of PCR primers is designed to identify whether the Sikowski vector is at the genomic locus. In this case, one primer binds to the genomic DNA and the second primer binds to the Sikowski vector.
• Typically, 3 transformants are screened per desired strain. Re-streak each colony onto the appropriate selection plate.
• 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.
• 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
    
• 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.
• 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

• Sikorski vector to be integrated	allele primers	vector primers
  pRS304*(TRP1)	DL11, DL17	DL11, DL22
  pRS305 (LEU2)	DL28, DL29	DL28, DL30
  pRS306 (URA3)	IP108, IP111	IP108, DL18

  • 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
  • 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.
  • Use the following table to determine whether the integration was successful.

  • gene to be integrated	size pcr product from allele primers	vector primers
    TRP1	435	1031
    trpD63	none	none
    LEU2	1168	1464
    leu2D1	none	979
    URA3	670	none
    ura3-52	699	651