((응답)) yeast subculture media

[colony]

		YEAST MEDIA Notes: All solid media should have 1.8 to 2.0% agar. Cool medium before adding tryptophan (TRP) or antibiotics. 1% amino acid solutions (ade, ura, his and leu) can be left at room temp. TRP should be kept in the fridge and cover bottle with foil. Do not autoclave TRP. Media with glucose (esp. YPD) can be overcooked. Avoid a dark brown color. Usually 25 min in the autoclave is sufficient and remove medium soon after pressure is down. Autoclaves stay hot if door is kept closed. Amino acid powder can be sterilized by boiling it in 2xSD for 2 min in microwave. 300 ml of agar takes approx. 2 min to melt in microwave. 4% agar can biol over easily. Use 3% for standard plates/4% for dissection plates. Agar can be over-cooked and weakened. Do not autoclave longer than 30 min if possible. Unsterilized Eppendorf tubes are fine to use for routine DNA work but not for yeast cultures that require sterility. Bubbles in media can be removed by spraying inside bottle with ethanol. No need to flame bottle after this step. Careful, people have been known to burn off their eyebrows. YPD (standard yeast complete medium) Lab-specific color marking on plates - RED stripe 10 g/l yeast extract 20 g/l bactopeptone 20 g/l glucose 18 g/l agar (you may wish to add 40 mg/l tryptophan and adenine to make up for deficiences in the medium) Using lab stock solutions: Melt 3% agar in microwave on Hi for about 3 min. Loosen cap. It tends to boil over. Need extra care of using 4%. Add 200 ml of 2 x YEP (yeast extract/ bactopeptone). Add 20 ml of sterile 40% glucose solution. Mix well. 20ml glucose is standard. However, 25 ml will allow the yeast to grow faster and survive longer in storage. Sporulation medium (see separate method site for sporulation protocol) Color marking on plates - two BLUE stripes 20 g/l Potassium acetate (KoAc) 0.2 g/l glucose Pre-grow the yeast in rich medium(e.g. YPD) to log phase first and aerate well. Then shake at room temp for 1 day (if possible) then 3- 4 days at 30 degreesC with shaking. Minimal (or defined) medium Color marking on plates - two BLACK stripes if no amino acids added 20 g/l glucose 1.67 g/l yeast nitrogen base without aminoacids or nitrogen. 5 g/l ammonium acetate 18 g/l agar 3.3 g/l of amino acid mix powder. Add 10 ml of sterile 1% amino acid solutions after cooling. Using lab stock solutions: Melt 3% agar solution in microwave for about 4 min. Loosen cap. It tends to boil over. Add 1.8g of amino acid powder to 200 ml of 2 x SD (yeast nitrogen base + N-source). Heat in microwave for 2 min until dissolved. Add to agar. Add 20 ml of sterile 40% glucose solution. Mix. Cool to below steaming and add additional amino acids from 1% stock solutions. Mix well, remove bubbles with ethanol spray bottle. Pour. Additional plate markings for defined media: - ADE double RED stripe - LEU single PURPLE stripe - HIS single BLACK stripe - TRP single GREEN stripe - URA single BLUE stripe Amino acid powder 2 g of each amino acid (except 4 g of Leucine). Uridine dissolves better than uracil. Leave out typical auxotrophies such as tryptophan, leucine, uracil/uridine, adenine, histidine. Mix powder well. YPGalactose (YPGal) or YPRaffinose (YPRaff) Color marking on plates (one and two ORANGE stripes respectively) 10 g/l yeast extract 20 g/l bactopeptone 50 ml/l of a 40% sterile FILTERED galactose or raffinose solution Raffinose dissolves slowly (you may wish to add 40 mg/l tryptophan and adenine to make up for deficiences in the medium) 18 g/l agar Using lab stock solutions: Melt 3% agar solution in microwave for about 4 min. Loosen cap. It tends to boil over. Add 200 ml of 2 x YEP (yeast extract/ bactopeptone). Add 25-30 ml of sterile 40% GAL or RAFF solution YEPglycerol (use to screen out petites – they wont grow using this non-fermentable C-source) Color marking on plate - RED/GREEN 10 g/l yeast extract 20 g/l bactopeptone 30 ml/l sterile filtered or autoclaved glycerol 18 g/l agar (you may wish to add 40 mg/l tryptophan and adenine to make up for deficiences in the medium) Using lab stock solutions: Melt 3% agar solution in microwave for about 4 min. Loosen cap. It tends to boil over. Add 200 ml of 2 x YEP (yeast extract/ bactopeptone). Add 30 ml of sterile 100% glycerol solution (or 60 ml of a 50% stock solution). Geneticin (G418) plates. Make YPDagar. While hot but not steaming , pour medium into 50 ml tube. Quickly add G418 to final concentration of 300 micrograms per ml. Mix well, remove bubbles with spray bottle. Pour 2 plates. Pour unused medium as YPD plates before medium sets. Store plates for 1 week at 4 degrees. G418 is kept at -20 as a 20 mg/ml in H2O solution. Not "kanamycin". Transformed cells require overnight in 2 ml YPD before plating.