ࡱ> a bjbj** .,H=bH=bR448\6tt(www$.^wwwwwwwpjqR06B B &B wwwwwwwwww6wwwwB wwwwwwwww4B v: BY4741 sporulation Friday: Mix cells of tester and library strain in a patch on a YPD plate for 3-6 hours then restreak on a selective plate and grow at 30 (selection is lys met for normal BY4741 x BY4742 mating) Monday, Tuesday, Wednesday: Patch a single colony onto a YPD plate overnight, then patch again on GNA overnight, and again on GNA overnight, all at 30( Thursday: Inoculate ~1 colony-worth of cells into 2.5ml of GMM media in a 14ml tube, vortex (OD should be ~0.1-0.5), put in 25 incubator shaking at 200rpm. Make sure tube is at a shallow angle like this:  Sporulate at 25( for 3-10 days (check them under the microscope every couple of days and dissect when you have plenty of tetrads) For tetrad dissection: Dry some YPD plates for a few days on the bench Mix 3l sporulated culture, 27l Buffer A (1.2M sorbitol, 50mM EDTA), 1l 17U/l high grade lyticase Incubate 30min at RT, then streak 3l over the appropriate area of the YPD plate Wait for the spots to dry then dissect immediately Media: GNA Presporulation Plates Final Concentrationsrecipe for 2 platesrecipe for 10 plates5% D-glucose 5 gm25 gm3% nutrient broth (powder or microgranulated)3 gm15 gm1% yeast extract 1 gm5 gm2% agar2 gm10 gmwater to the final volume100 ml500 mlAutoclave for 15 minutes. Plates should always be freshly made and last only for 1-2 weeks at 4C for optimum sporulation. Liquid Sporulation Medium Final ConcentrationsStock solutionsrecipe for 20 mls1% Potassium Acetate10% Potassium Acetate (20g/200ml)2 mls0.005% Zinc Acetate0.5% Zinc acetate (1g/200ml)0.2 mlsInclude amino acids/nucleotides from 100x / 10x stocks to complement any homozygous auxotrophies in the diploid. Not doing this can reduce spore viability substantially. 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