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Strain Verification

For each ORF, two independent deletion mutants must be produced.
This can be done in either of three ways:

Haploid transformants:
A-KanB and KanC-D tests must be positive with the deletion mutant and negative with wild-type. If one of these reactions doesn't work, the A-D* test can substitute.
A-B and C-D tests: Both must be negative with the deletion strain and positive with wild-type.

Heterozygotes:
A-B, C-D, A-KanB and KanC-D PCR products must be present. An A-D* test can substitute if either the A-KanB or KanC-D PCR doesn't work.* Because the sizes of the A-D bands can be similar in wild-type and mutant strains, it can be helpful to digest part of the A-D reaction with HindIII to test for the HindIII site in the KanMX4 cassette

Haploid segregants:
A-B and C-D products must be present in wild-type but not in the mutant. For at least one deletion-specific primer pair, either an A-KanB, KanC-D, or an A-D band of the expected size must be present.

Homozygous diploids:
Must be met+lys+, streaked to single colonies, and tested for both
mating and sporulation.

Additional procedures for verifying diploid strains*

1. From mating, select MET+ LYS+ progeny
      If none:
        a) Check met, lys and MAT markers of haploid parents
               (because one haploid could be Met- Lys-.)
        b) If markers okay, check mating visually.
               If no mating observed, then:
                  Record that no homozygous diploid could be obtained as 'no reason                   unknown', Comment 'possible bilateral mating defect'
              (Note: it would have to be a bilateral defect, since the haploids mated when you                determined that they were a and alpha using testers.)

3. Pick one 'diploid' and check for mating to a and alpha testers.
Include positive and negative controls on test plates!

If no mating: save as your homozygous diploid.



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		Strain Verification For each ORF, two independent deletion mutants must be produced. This can be done in either of three ways: Transformation of haploids. The diploids are then obtained by mating. Transformation of diploids. The haploids and homozygous diploids are obtained by sporulation and mating Transformation of one of the haploid strains and the diploid strain. The other haploid strain is obtained from the diploid by sporulation and the homozygous diploid is made by mating of the two haploids. The genotypes of the a and alpha segregants have to match those of BY4741 and BY4742, respectively. Haploid transformants: A-KanB and KanC-D tests must be positive with the deletion mutant and negative with wild-type. If one of these reactions doesn't work, the A-D* test can substitute. A-B and C-D tests: Both must be negative with the deletion strain and positive with wild-type. Heterozygotes: A-B, C-D, A-KanB and KanC-D PCR products must be present. An A-D* test can substitute if either the A-KanB or KanC-D PCR doesn't work.* Because the sizes of the A-D bands can be similar in wild-type and mutant strains, it can be helpful to digest part of the A-D reaction with HindIII to test for the HindIII site in the KanMX4 cassette Haploid segregants: A-B and C-D products must be present in wild-type but not in the mutant. For at least one deletion-specific primer pair, either an A-KanB, KanC-D, or an A-D band of the expected size must be present. Homozygous diploids: Must be met+lys+, streaked to single colonies, and tested for both mating and sporulation. Additional procedures for verifying diploid strains* 1. From mating, select MET+ LYS+ progeny If none: a) Check met, lys and MAT markers of haploid parents (because one haploid could be Met- Lys-.) b) If markers okay, check mating visually. If no mating observed, then: Record that no homozygous diploid could be obtained as 'no reason unknown', Comment 'possible bilateral mating defect' (Note: it would have to be a bilateral defect, since the haploids mated when you determined that they were a and alpha using testers.) 2. Streak on SC-met-lys for single colonies. 3. Pick one 'diploid' and check for mating to a and alpha testers. Include positive and negative controls on test plates! If no mating: save as your homozygous diploid. If apparently mating to one tester: a) Check met, lys and MAT markers of haploid parents. (One haploid could be Met+ Lys+) b) If markers okay, pick other 'diploids' to test. c) If all 'diploids' act the same, comment 'possible chromosome instability.' d) Do sporulation check. If sporulation check indicates diploid, save as your homozygous diploid. If not, try calcofluor staining for bud sites. If apparently mating to both testers: a) check met, lys and MAT markers of haploid parents. (Haploids could be Met+ Lys+.) b) If markers okay, pick other 'diploids' to test. c) If all 'diploids' act same, comment 'possible chromosome instability.' d) Do sporulation check. If sporulation check indicates diploid, save as your homozygous diploid. If not, try calcofluor staining for bud sites * From Petra Ross-Macdonald