Yeast Gene Deletion/Disruption Method From Chandra Theesfeld and Daniel Lew, Duke University Medical Center Method can be usd to generate a PCR fragment with ends homologous to the gene to be disrupted flanking a selectable marker (HIS3, LEU2,TRP1, or URA3 from the pRS vectors of Sikorski and Hieter, 1989). Forward primer: 5' YFG (40-50 bases) - CGTTTCGGTGATGAC 3' (base number 6 through 20 in pRS vectors) Reverse primer: 5' YFG (40-50 bases) - TTCCTGATGCGGTATTTTCTCCT 3' (common sequence of vector pRS flanking the selectable marker) pRS303/313 (HIS3): bases 1424-1401 (PCR product of ca. 1500 bp) pRS304/314 (TRP1): bases 1238-1215 (PCR product of ca. 1300 bp) pRS305/315 (LEU2): bases 2475-2452 (PCR product of ca. 2550 bp) pRS306/316 (URA3): bases 1344-1321 (PCR product of ca. 1400 bp) Use standard 20-25 µl PCR reaction mixtures (with Taq polymerase) to generate the disruption fragments (enough for at least four yeast transformations). 94C, 4 min 1 cycle 94C, 1 min 52C, 45 sec 72C, 2 min 35 cycles 72C, 4 min 1 cycle 4C hold Confirm gene disruptants by PCR analysis of transformants' DNA, using one of these primers and a third primer in the gene outside the disruption fragment. Generally, about 20% of the transformants have gene disruptions, comfirmed by PCR. YFG = Your Favorite Gene