TEWLAB Yeast Media - Recipes
All media and components should be labeled precisely, including your initials and the date.
CARBON SOURCE STOCKS
1) 20% Dextrose (= 10X).
a) Put 100 g Dextrose into a 500 ml bottle.
b) Add 500 ml 1X-distilled water. Do this quickly and swirl it and the Dextrose will
dissolve quickly, but don't worry if it doesn't all dissolve, it will do so in the
autoclave.
c) Autoclave 20 min.
2) 20% other sugars (= 10X). Sugars other than glucose will break down if autoclaved, and
so they are always sterile filtered.
a) Put 100 g of the sugar into a beaker (or scale down as needed).
b) Add ~400 ml 1X-distilled water and heat and stir until dissolved. Many sugars
dissolve very slowly. Add more water if needed, but don't go over 500 ml.
c) Bring to 500 ml in a graduated cylinder.
d) Sterile filter into a sterile 500 ml bottle. Store at RT.
NUTRIENT SOURCE STOCKS
1) YP. See YPA in MEDIA, below.
2) YNB + CSM mixes (= 10X). These stocks are prepared as a combination of the nitrogen
base (i.e. YNB + AS) plus the desired CSM mix. Thus, a typical SD medium is made by
simply adding a carbon source and one of these mixes. These stocks should be labeled
"YNB + CSM – nutrients". We typically do not write the "AS", but you should write the
"YNB" to be clear that it was added.
a) Weigh out 33.5 g of YNB + AS. If you are not certain you have the right bottle,
ASK!
b) Also weigh out the appropriate amount of the desired CSM mix to make 500 ml
of a 10X solution. The bottles are labeled with the g/liter of a 1X solution. So,
this number should be multiplied by 5. For example, CSM is used at final 0.79
g/liter. 0.79 x 5 = 3.95 g. The mixes are all different because they contain
different combinations of nutrients.
c) Add the above to 500 ml 1X-distilled water and heat and stir until ~dissolved.
You will need to heat it to about 80 C, but do not let it boil. The CSM has a
tendency to float; make sure it gets down into the water.
d) After heating, transfer to a stir plate without heating and stir an additional 20-30
minutes to cool and dissolve completely. Note that there is always a small
amount of particulate matter that remains – don't worry about this.
e) Sterile filter into a sterile 500 ml bottle. Store wrapped in foil at 4 C. When
making more than one CSM mix, you should use the same filter if possible, but
being sure not to cross-contaminate. For example, CSM – Leu – Ura could be
filtered first, followed by CSM – Ura (but not vice versa – think about it!).
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NUTRITIONAL SUPPLEMENT STOCKS
1) CSM. Prepared as a mixed stock with the YNB + AS, see above.
2) Individual Nutrients (= 100X). Simply dissolve in water at 100 times the concentration
listed in the Bio101/Qbiogene catalog or in TEWLAB Yeast Media – General
Description. Put either into a regular bottle, or into the brown dropper bottles, and
autoclave 20 min. Store at RT.
ADDITIVE STOCKS
1) G418 (= 100X). This is tricky because G418 powder is not 100% active. You need to
make a stock solution of 20 mg/ml ACTIVE G418. The active fraction is listed on the
bottle, but it changes with every bottle. So, if you want to make 10 ml of stock and the
bottle says 0.727 for the fractional activity, you need (10 ml x 20 mg/ml)/0.727 = 275 mg
of G418. After weighing, dissolve in sterile distilled water. Aliquot as convenient and
store at –20 C.
2) Canavanine (= 100X). Dissolve canavanine at 5 mg/ml in sterile distilled water. Aliquot
as convenient and store at –20 C.
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MEDIA
General comments on plate pouring:
a) AGAR MEDIA MUST BE THOROUGLY MIXED!!
b) Your goal is to get 38-40 plates per liter of medium. Less than 35 plates per liter will be
way too thick; greater than 40 plates per liter are too thin. Work to achieve plates that are
of uniform thickness and level.
c) If you have a lot of plates to pour, media can be kept in the water bath at 55 C to prevent
it from hardening, but this is not generally necessary.
d) Agar media can be prepared in any suitable glassware. People have different preferences.
Some always use Erlenmeyers, I personally prefer 500 ml bottles.
e) Label plates with appropriate color codes from database BEFORE pouring.
f) After pouring your plates, immediately and thoroughly rinse out the glassware to prevent
agar from hardening inside it!
g) Pouring plates can be done however is most comfortable for you. I prefer to pour in
stacks of five, pouring the bottom plate first, and on up the stack.
h) Bubbles on the surface of the agar are removed after pouring by lightly passing over them
with a Bunsen burner. But don't melt the plastic!
i) Once plates are hardened they should be inverted and allowed to air dry for 2-3 days prior
to bagging and storing at 4 C.
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YPA. YPAD and other YPA-based media are made as a YPA stock, to which Dextrose or other
carbons sources are added after autoclaving. Because the carbon sources are almost all made as
10X stocks, this dictates that the YPA stock is made up in 90% of the final volume.
a) Measure ingredients according to the table (those above the line only).
b) Add the indicated amount of water. Do not worry about dissolving the solids –
this will happen in the autoclave. For agar plates, many people choose to add a
stir bar prior to autoclaving for mixing after autoclaving.
c) Autoclave 20 min.
d) For liquid YPA, bottles are simply placed on the shelf – carbon sources and
additives are added by the user before inoculation.
e) For plates, add carbon source and additives, MIX WELL, and then pour plates.
See general comments above.
Component
0.5 liter
1 liter
2 liter
Yeast extract
5g
10 g
20 g
Bacto-Peptone
10 g
20 g
40 g
(Agar - plates only)
(10 g)
(20 g)
(40 g)
100X Adenine
5 ml
10 ml
20 ml
1x distilled water
450 ml
900 ml
1800 ml
Carbon source
(added after autoclaving)
50 ml
100 ml
200 ml
Additives
(added after autoclaving)
As needed
As needed
As needed
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Synthetic Defined (SD). Liquid SD medium is made by simply mixing the needed combination
of YNB + CSM stock and carbon source stock, as well as any additional supplements or
additives, in a sterile tube or bottle, and then bringing to volume with sterile distilled water. SD
plates are made by adding the same components to molten agar, as follows:
a) Weigh the agar according to the following table.
b) Add the indicated amount of water. Many people choose to add a stir bar prior to
autoclaving for mixing after autoclaving.
c) Autoclave 20 min.
d) Add carbon source, CSM mixes and additives, MIX WELL, and then pour plates.
See general comments above.
Component
0.5 liter
1 liter
2 liter
Agar - plates only
10 g
20 g
40 g
1x distilled water
400 ml
800 ml
1600 ml
Carbon source
(added after autoclaving)
50 ml
100 ml
200 ml
YNB + CSM mix
(added after autoclaving)
50 ml
100 ml
200 ml
Additives
(added after autoclaving)
As needed
As needed
As needed
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Preparation of 5-FOA plates (1 liter)
1) 20g agar
400ml ddH20
Autoclave the above mixture
2) 1g of 5-FOA in 400ml ddH2O
-Dissolve in stirring with moderate heat “4” (This takes several hours)
-Filter sterilize
3) 100ml of sterile 20% glucose
4) 100ml of 10X CSM-complete stock
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