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Mapping strains: chromosome loss mapping with CD6 (AB1)

lys7 mutants are temperature-sensitive.

Temperature: 25.0°C

Atmosphere: Typical aerobic

Protocol: The basic procedure for inducing chromosome loss is: 1) incubate a cdc6/cdc6 multiply-heterozygous diploid (3000 cells/plate) on YEPD for 6 hours at 35C, 2) shift to 23C, and 3) grow the survivors (about 300 cells) at 23C for a few days.Replica plate the surviving colonies onto diagnostic media, paying particular attention to the very small colonies with long lag periods before growth at 23C. Usually 10 YEPD plates will be adequate; however, because of differences in viabilities among strains, plates with different numbers of starting cells may be required.For a dominant unmapped marker, X, cross the mutant strain to cdc6 his4. Sporulate and dissect the diploid in order to place the marker into a cdc6 haploid background. Mate the cdc6 X strain to N439-. Loss of the chromosome carrying X should uncover a recessive mapped marker on the homolog.For a recessive unmapped marker, y-, cross the mutant to N439-. Sporulate and dissect the diploid. Pick up cdc6 y- haploids in a variety of multiply auxotrophic backgrounds or backcross to N439- to get the recessive marker in a strain marked on all chromosomes. Mate cdc6 y- to a cdc6 his4 haploid. Loss of the chromosome carrying Y should uncover other recessive markers on the homolog.

Protocol: The basic procedure for inducing chromosome loss is: 1) incubate a cdc6/cdc6 multiply-heterozygous diploid (3000 cells/plate) on YEPD for 6 hours at 35C, 2) shift to 23C, and 3) grow the survivors (about 300 cells) at 23C for a few days.Replica plate the surviving colonies onto diagnostic media, paying particular attention to the very small colonies with long lag periods before growth at 23C. Usually 10 YEPD plates will be adequate; however, because of differences in viabilities among strains, plates with different numbers of starting cells may be required.For a dominant unmapped marker, X, cross the mutant strain to cdc6 his4. Sporulate and dissect the diploid in order to place the marker into a cdc6 haploid background. Mate the cdc6 X strain to N439-. Loss of the chromosome carrying X should uncover a recessive mapped marker on the homolog.For a recessive unmapped marker, y-, cross the mutant to N439-. Sporulate and dissect the diploid. Pick up cdc6 y- haploids in a variety of multiply auxotrophic backgrounds or backcross to N439- to get the recessive marker in a strain marked on all chromosomes. Mate cdc6 y- to a cdc6 his4 haploid. Loss of the chromosome carrying Y should uncover other recessive markers on the homolog.

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