FANDOM



This will tell you how to make; Basic, -lys, -leu, -met, -ura, -his, G418, 5-FOA, and YPD Edit

All Yeast Plates follow the same basic principle however the ingredients needed for each plate varies.

  1. Add Stir Bar to appropriately sized flask (eg. for 1L of media use a 2L flask) and add 200 mL of ddH20 for every liter of media which you are making.
  2. Mix all of your ingredients save for your glucose and any drugs you may be adding to the plate.
  3. Bring solution to desired total volume
  4. When needed, bring the pH of your media to your desired pH (pH the C- plates to 5.9).
  5. Cover your flask with new aluminium foil and fresh autoclave tape
  6. AUTOCLAVE
  7. After autoclaving add the appropriate amount of 50% Glucose (to 2%), or 80% glycerol for YPG (to 3%) and after allowing the media to cool to the point at which you can hold your hand to the flask, add any drugs you need
  8. Pour plates (25mL per plate) on a sterilized surface (eg. cleaned with 95% EtOH, and after it has dried lighting a Bunsen Burner), Should bubbles form on the plate while pouring either suck the bubbles back up or flame them. WATCH SOMEONE DO THIS BEFORE YOU ATTEMPT TO DO SO YOURSELF!
  9. Let the plates cool and solidify overnight or for a day, and label them with the correct color and name before bagging them in the sterile plastic bags provided to us. (you can find the color code here)
  10. Place your plates on the appropriate shelves in the back of the Kennedy Lab (near the Thermo Cyclers) or if they have drugs in them, place them in 4C cold room.

Here is a table of Ingredients for each plate type Edit

Plate Type Color Code

Stripe

Ingredients Volume for 1 L Special Notes
YPD Black
  1. Peptone
  2. Yeast Extract
  3. Agar
  4. Glucose (50% w/v stock)
  1. 20g
  2. 10g
  3. 20g
  4. 40ml
  • Remember to add Glucose AFTER autoclaving

See Link for instructions

Basic Brown
  1. Yeast Nitrogen Base w/out amino acids and W/out ammonium Sulfate
  1. Ammonium Sulfate
  2. Bacto Agar
  3. Glucose (50% w/v stock)
  1. 1.7g
  2. 5g
  3. 17g
  4. 40mL
  • Add Glucose AFTER autoclaving.
  • Bring pH to 5.9 BEFORE autoclaving.
  • Link to how to use pH meter
C-URA Blue
  1. Yeast Nitrogen Base w/out amino acids and W/out ammonium Sulfate
  1. Ammonium Sulfate
  2. Bacto Agar
  3. C-URA Amino Acid Powder
  4. Glucose (50% w/v stock)
  1. 1.7g
  2. 5g
  3. 17g
  4. 1.4g
  5. 40mL
  • Remember to add Glucose AFTER autoclaving.
  • Bring pH to 5.9 BEFORE autoclaving
  • Link to how to use pH meter
  • Amino Acid powder can be found in the autoclave room on the first floor (if not in the dark room).
  • L-URA isn't the same as C-URA (L-stands for its optical rotation while C-stands for complete)
C-LEU Red
  1. Yeast Nitrogen Base w/out amino acids and W/out ammonium Sulfate
  1. Ammonium Sulfate
  2. Bacto Agar
  3. C-LEU Amino Acid Powder
  4. Glucose (50% w/v stock)
  1. 1.7g
  2. 5g
  3. 17g
  4. 1.4g
  5. 40mL
  • Add Glucose AFTER autoclaving.
  • Bring pH to 5.9 BEFORE autoclaving
  • Amino Acid powder can be found in the autoclave room on the first floor (if not in the dark room).
  • L-LEU isn't the same as C-LEU (L-stands for its optical rotation while C-stands for complete)
C-LYS Yellow
  1. Yeast Nitrogen Base w/out amino acids and W/out ammonium Sulfate
  1. Ammonium Sulfate
  2. Bacto Agar
  3. C-LYS Amino Acid Powder
  4. Glucose (50% w/v stock)
  1. 1.7g
  2. 5g
  3. 17g
  4. 1.4g
  5. 40mL
  • Add Glucose AFTER autoclaving.
  • Bring pH to 5.9 BEFORE autoclaving.
  • Amino Acid powder can be found in the autoclave room on the first floor (if not in the dark room).
  • L-LYS isn't the same as C-LYS (L-stands for its optical rotation while C-stands for complete)
C-MET Orange
  1. Yeast Nitrogen Base w/out amino acids and W/out ammonium Sulfate
  1. Ammonium Sulfate
  2. Bacto Agar
  3. C-MET Amino Acid Powder
  4. Glucose (50% w/v stock)
  1. 1.7g
  2. 5g
  3. 17g
  4. 1.4g
  5. 40mL
  • Bring pH to 5.9 BEFORE autoclaving.
  • Add Glucose AFTER autoclaving.
  • Amino Acid powder can be found in the autoclave room on the first floor (if not in the dark room).
  • L-MET isn't the same as C-MET (L-stands for its optical rotation while C-stands for complete)
C-HIS Green
  1. Yeast Nitrogen Base w/out amino acids and W/out ammonium Sulfate
  1. Ammonium Sulfate
  2. Bacto Agar
  3. C-HIS Amino Acid Powder
  4. Glucose (50% w/v stock)
  5. C-HIS Amino Acid Powder
  1. 1.7g
  2. 5g
  3. 17g
  4. 1.4g
  5. 40mL
  • Add Glucose AFTER autoclaving.
  • Bring pH to 5.9 BEFORE autoclaving.
  • Amino Acid powder can be found in the autoclave room on the first floor (if not in the dark room).
  • L-HIS isn't the same as C-HIS (L-stands for its optical rotation while C-stands for complete)
G418 Purple
  1. Peptone
  2. Yeast Extract
  3. Agar
  4. Glucose (50% w/v stock)
  5. G418 (100mg-ml stock)
  1. 20g
  2. 10g
  3. 20g
  4. 40ml
  5. 2ml
  • Add Glucose after autoclaving
  • Add G418 when the media is about 50C (can hold your hand to the flask for 5 sec)
  • Store in 4C cold room when solid
YPG Black and Red
  1. Peptone
  2. Yeast Extract
  3. Agar
  4. Glycerol (80% w/v stock)
  1. 20g
  2. 10g
  3. 20g
  4. 37.5ml
  • Remember to add Glycerol AFTER autoclaving
5-FOA Orange and Green
  1. Agar
  2. Ammonium Sulfate
  3. Yeast Nitrogen Base (YNB) w/o amino acids
  4. His, Leu, and Lys powder
  5. Ura powder
  6. Glucose (50%)

---------------------

In a separate 500 mL beaker:

  1. ddH2O
  2. 5-FOA
  1. 20g
  2. 5g
  3. 5g
  4. 1.4g of each
  5. 20mg
  6. 40mL

-------------------

  1. 210mL
  2. 1g
  • Before autoclaving the agar, use the pH meter to bring the solution to pH = 5.8.
  • As always, add the Glucose after autoclaving.
  • Add the 5-FOA after autoclaving the agar.
  • You must filter sterilize the 5-FOA solution.

See link for full instructions.

Here is a list of Part number Edit

Name Brand/Part Number
Peptone BD 211677
Yeast Extract BD 212750
Agar BD 214010
Glucose Sigma 59378 or Bio-World
Ammonium Sulfate SPECTRUM AM185
yeast nitrogen base without amino acids and ammonium sulfate BD 233520
100X15mm plates. BD Falcon 351029