Generation and analysis of haploid deletion mutants
1) Transformation procedure
48 heterozygous diploid strains were patched from glycerol stocks on to YE agar supplemented with uracil (ura) and leucine (leu) but no adenine (ade) + G418 (Duchefa Biochemie) (YEG) in two 96 well microtitre plates (so that each strain was represented 4 times) and left to grow for 2-3 days at 32°C. Any strains that did not grow under these conditions were repeated later on YE (no ade, no G418). Any strains that were pink or red were not used as these may be haploids. Cells were inoculated into 200 ?l YEG + ade, leu, ura (YEGS) and left to grow into stationary phase. Ade, ura and leu were all added at 250 mg/L, and G418 was added at 100 ?g/ml. The strains were transformed using a modified version of the PLATE method7 as follows.
1) Spin down at 2,500rpm for 3 min in Beckman GS-6KR, remove supernatant, and resuspend cells in 20 μl of water.
2) To each well, add 20 μg herring sperm DNA and ~1.5 μg of pON177 DNA and 200 μl PEG/LiAC/TE [40% PEG4000 in 1X TE/LiAc (0.1 M LiAC, 10 mM Tris/pH7.5, 1 mM EDTA) and filtering to sterile], and mix well. (40% PEG = 20 g in 50 ml TE/LiAc, pH 7.5) pON 177 was a gift from Olaf Nielsen, which is based on pIRT1 (Kelly 1988), a URA3-based plasmid containing the 9 kb SalI-SphI fragment containing the mat1-M cassette. This cassette has 263-bp deletion (smt-0 mutation that prevents switching but does not affect expression of the M mating type).
3) Incubate for 3 days in 25°C incubator. Seal the lid to prevent evaporation.
4) Spin down at 2,500prm for 3min in Beckman GS-6KR and remove supernatant.
5) Wash with 200 ml water, spin and resuspend in final volume of 100 ml water
6) Using 48 minimal + leu (ML) plates (2 for each column of each 96-well plate) pipette 2.5 μl X 16 for each strain (8 spots /plate).
7) Incubate the plates at 32°C for several days as it may take a week for colonies to appear.
8) Inoculate a transformant from each strain (i.e., 4 transformants for each deletion) into minimal with NH4Cl replaced by 1mg/ml glutamic acid + ade, leu (-N+S) in the 96-well microtitre plates, and incubate at 25°C for 2~3 days.
9) Spin down and resuspend in 200 μl of 1 X helicase (1 in 50 dilution from the Bio Sepra). Incubate overnight at 25°C.
10) Check several wells to see that the vegetative cells are digested and that spores are present. Spin down and resuspend in 200 μl water. The spores can be left at 4°C for a week or two.
2) Screening haploid deletion strains
1) Spores were diluted 1in 50 and 5 μl of each strain was plated on to YE + ade/leu/ura (YES) agar, so that each 9 cm petri dish had 4 isolates for each of 8 different deletion strains.
2) Screen germinating spores for the deletion phenotype on 32°C plates after one day.
3) Screen for the deletion phenotype at 32°C and 25°C after 2 days, the replica plate on to YEGS agar, and incubate at 25°C and 32°C.
4) Screen YEGS agar plates at 25°C and 32°C after one day and 4 days to confirm the deletion phenotype.
5) Streak out viable haploids on YEGS agar at 32°C (or 25°C, if ts), recheck the phenotype, and test phenotype at 36°C. Strains were also replica plated to minimal + ade, leu to check cells no longer contained pON177 and to YE + leu, ura to check the ade6 allele. Haploids of genotype (ade6-M210, leu1-32, ura 4-D18 and ade6-M216, leu1-32, ura 4-D18) were selected and glycerol stocks prepared.
Note: About 5% of the heterozygous deletion diploids could not be transformed using this high throughput method. These strains were repeated using a standard transformation protocol4.
3) Tetrad analysis
Biological repeats of all essential genes from the random spore analysis were carried out and analysed by tetrad analysis. Cells were dissected using a Singer MSM machine on YES plates. Spores were left to germinate for 4~5 days and the essentiality determined. Viable colonies were patched onto YES plates containing 100 μg/ml G418 to confirm that viability was associated G418 sensitivity.