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. Transfer 5.5 uL of the mixture into a second PCR tube.
• 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
• Add 45 uL PCR mix to each tube. For each colony, you will have two PCR reactions corresponding to the two sets of PCR primers. The PCR mix 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 at locus	pcr product size
	allele primers	vector primers
TRP1	435	1031
trpD63	none	none
LEU2	1168	1464
leu2D1	none	979
URA3	670	none
ura3-52	699	651

Archiving Yeast Strains

• Incoculate YEP media with a single yeast colony.
• Grow overnight at 30C in a roller drum.
• Mix 50% glycerol and yeast culture in a 2 mL screw-top vial to give a final glycerol concentration of 15%(v/v), e.g. 300 uL 50% glycerol and 700 uL yeast culture.