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. Author manuscript; available in PMC: 2014 Aug 12.
Published in final edited form as: Mol Cell. 2008 Apr 25;30(2):248–258. doi: 10.1016/j.molcel.2008.02.021

Figure 1. Diploid Shuffle—A Method for Generating ts Alleles for the Essential Genes in Saccharomyces cereviviae.

Figure 1

(A) Genomic DNA containing YFEG and its 5′ and 3′ regions is used as the template for PCR mutagenesis. Two black horizontal arrows represent the gene-specific primers used. The mutagenized PCR product is cloned into the vector SB221+ Topo-TA (mutations are represented by black stars). The Topo-TA cloning site is represented by a red T, and the A overhang protruding from the PCR product is represented by a red A. Left beige bar represents the 5′ half of the KanMX selectable marker (Kan), while the right beige bar represents the other half of the KanMX selectable marker (MX). The NotI restriction sites are indicated by two diagonal black arrows.

(B) The product of the cloning step is a library of a mutagenized YFEG. The library is then transformed into E. coli and digested with NotI to release linear fragments (following DNA purification).

(C) The linearized library is transformed into the corresponding heterozygous diploid strain. Blue and red bars that flank the KanMX knockout represent the two bar codes.

(D) Heterozygous diploid transformants are sporulated (following meiosis), and MATa Ura+ haploids spores are selected on haploid-selective medium at 25°C.

(E) Selection of ts candidates following the replica plating and incubating at 25°C and 37°C. Black arrows identify a potential ts allele.