Notes for setting up Neurospora lab for tryptophan/nicotinic pathway.

Strains used Stock number
Wild type FGSC # 988
trp-1 (allele 10575) FGSC # 4049
trp-2 (allele 10) FGSC # 1850
trp-3 (allele td101)  FGSC # 1010
trp-4 (allele Y2198) FGSC # 4059
nic-1 (allele S1413) FGSC # 763
nic-2 (allele 43002) FGSC # 2527
nic-3 (allele Y31881) FGSC # 2528
nic-1; nic-2 FGSC #8407
nic-1; nic-3  FGSC #8408
nic-2; nic-3 FGSC #8409

How to store cultures:
Slants of Vogel medium with 1mM tryptophan and 2 ug/ml of nicotinamide, 2% sucrose. Grow at Room temp., let conidiate abundantly, then store at -20 C. Should stay alive at least 10 years. There is no point in freezing a slant that has not conidiated because mycelial cultures are 100% killed by freezing.

To prepare conidia for class: put 20 ml trp agar (1.5% agar) or nic agar, as above, into 125 ml flasks plugged with cotton, not foil. Let dry at room temp, out of bright light for one week or until there is no more condensation on walls of flasks. Inoculate and grow at room temp, but not above 25 C, in ambient of strong light, not in dark for 1-2 weeks.

To prepare 'spent medium' for cross feeding experiment:
Make up 1X Fries and add 1.5% sucrose and 0.1 mM tryptophan.
(Fries' medium is prererable to Vogel's medium for certain experiments because it has a much lower concentration of salts).

2000 ml 1X Fries

30 g sucrose

40 mg tryptophan

Aliquot 20 ml of the above solution into about 100 125 ml flasks, plug the opening with cotton and autoclave for 15 minutes. After the medium has cooled, inoculate about 1/3 of the flasks with wild type, 1/3 with trp-1 and 1/3 with trp-2 mutant spore suspensions. The cultures should grow without shaking for about 7 days at 25 C or room temperature in a lighted area. After growing, decant off the spent medium carefully, using a millipore filter to sterilize the medium. The spent medium can be stored indefinitely

Nutrients used in agar plates

ANTRHANILIC ACID- 20 mg/ml pH4 can be autoclaved, but comes out of solution in a few hours 0.5 g/25 mls

INDOLE.- 10 mg/ml of 50% ETOH. Sterilize by filtering. pH comes out OK at about 6.5. May precipitate upon addition to hot medium, shake well and pour plates anyway. The precipitate wil redissolve after the agar gels.

TRYPTOPHAN- 10 mg/ml or 0.25 g/ 25 ml pH OK. May not go into solution until autoclaved

HYDROXYANTHRANILIC ACID- 2 mg/ ml pH5. Will not go into solution until neutralized- add 1-2 drops 1N NaOH / 5 ml. Sterilize by filtration. Unstable, turning yellow. Store frozen.

QUINOLINIC ACID- 50 mg/ ml pH 1. Will not go into solution until neutralized- add about 15 drops 10 N NaOH / 5 ml. Sterilize by filtration

NICOTINAMIDE- 1 mg / ml pH 6.5 OK. autoclave to sterilize

HYDROXYKYNURENINE- 2.5 mg/ml adjust pH to neutral and filter sterilize

Vogels Medium N (50X stock)

In 750 ml. distilled water, dissolve successively with stirring at room temperature

Na3 citrate-5 1/2 H2O                 150 grams
KH2PO4, anhydrous                     250 grams
NH4NO3, anhydrous                     100 grams
MgSO4-7 H2O                           10 grams
CaCl2-2 H2O                           5 grams
Trace Element Solution (see below)     5 ml.
Biotin Solution (see below)            2.5 ml.

The Biotin solution is prepared by dissolving 5 mg Biotin in 100 ml of 50% (v/v) Ethanol

Trace Elements

The trace element solution (containing citric acid as a solubilizing agent) is made up as follows: In 95 ml. distilled water, dissolve successively with stirring at room temperature:

Citric acid-1 H2O         5. 00 grams
ZnSO4-7 H2O               5. 00 grams
Fe(NH4)2(SO4)2-6 H2O       1. 00 gram
CuSO4-5 H2O               0. 25 gram
MnSO 4-1 H2O              0. 05 gram
H 3BO 3, anhydrous         0. 05 gram
Na 2 MoO 4-2 H2O           0. 05 gram

The resulting total volume is about 100 ml. Chloroform (1 ml. ) is added as a preservative, and the trace element solution is stored at room temperature.

20 X Sugar solution (also called FGS)

Used in both the minimal and nutrient agar plates. Prepare about 3 liters.

20% sorbose 200 g
1 % glucose 10 g
1 % Fructose 10 g

Add to about 750 ml water, dissolve and adust volume to 1 L. Autoclave before formulating medium as the sugars will caramelize in the presence of salts

Minimal Agar plates

for 1000 ml volumes

Add 20 ml of Vogels 50X to 930 ml distilled water. Add 10 g Bacto Agar, Shake and autoclave. After the media comes out of the autoclave, sterilely add 50 ml of the 20 X sorbose sugar solution. Mix and pour plates

Nutrient agar plates

These are prepared the same way as the minimal plates except 5 ml of sterile nutrient / 1000 ml are added separately, in addition to the 20 X sugar solution, after the medium is autoclaved.

Return to the teaching page