6-Azauracil Sensitivity Assay for Yeast
William P. Tansey
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
Corresponding author (tansey@cshl.edu)
INTRODUCTION
Treatment of yeast with 6-Azauracil (6AU) leads to a reduction of intracellular GTP levels. The
reduction in GTP levels is not itself lethal, but can block yeast growth when combined with
mutations that affect transcriptional elongation. 6AU sensitivity thus can be used as a crude assay to
test for mutations that affect transcriptional elongation. The assay described here requires growing
saturated cultures of yeast, counting, and spotting serial dilutions of yeast on both CSM and CSM +
6AU plates.
MATERIALS
1.Reagents
1.1
6-Azauracil (6AU) (Sigma-Aldrich)
1.2
Ammonium hydroxide (1 M)
1.3
CSM for liquid yeast cultures
1.4
CSM/agar for 20 plates
1.5
TE buffer
1.6 Yeast cultures
1.1
6-Azauracil (6AU) (Sigma-Aldrich)
Azauracil is toxic and may be harmful by inhalation, ingestion, or skin absorption. Avoid contact with the solid or
solution. Wear appropriate gloves and safety glasses and use in a chemical fume hood.
1.2
Ammonium hydroxide (1 M)
Ammonium hydroxide (NH4OH) is a solution of ammonia in water. It is caustic and should be handled with great
care. As ammonia vapors escape from the solution, they are corrosive, toxic, and can be explosive. Use only with
mechanical exhaust. Wear appropriate gloves. Use only in a chemical fume hood.
1.3
CSM for liquid yeast cultures
Complete Minimal (CM) or Synthetic Complete (SC) and Drop-out Media
Bact-yeast nitrogen base without amino acids*, 6.7 g
glucose, 20 g
Bacto Agar, 20 g
#drop-out mix, 2 g1
H2O, to 1000 ml
To test the growth requirements of strains, it is useful to have media in which each of the commonly
encountered auxotrophies is supplemented except the one of interest (dropout media). Dry growth
supplements are stored premixed.
CM (or SC) is a medium in which the drop-out mix contains all possible supplements (i.e., nothing
is "dropped out").
*Yeast nitrogen base without amino acids (YNB) is sold either with or without ammonium sulfate.
This recipe is for YNB with ammonium sulfate. If the bottle of YNB is lacking ammonium sulfate,
add 5 g of ammonium sulfate and only 1.7 g of YNB.
1.4 CSM/agar for 20 plates
Complete Minimal (CM) or Synthetic Complete (SC) and Drop-out Media
Bact-yeast nitrogen base without amino acids*, 6.7 g
glucose, 20 g
Bacto Agar, 20 g
#drop-out mix, 2 g
H2O, to 1000 ml
To test the growth requirements of strains, it is useful to have media in which each of the commonly
encountered auxotrophies is supplemented except the one of interest (dropout media). Dry growth
supplements are stored premixed.
CM (or SC) is a medium in which the drop-out mix contains all possible supplements (i.e., nothing
is "dropped out").
*Yeast nitrogen base without amino acids (YNB) is sold either with or without ammonium sulfate.
This recipe is for YNB with ammonium sulfate. If the bottle of YNB is lacking ammonium sulfate,
add 5 g of ammonium sulfate and only 1.7 g of YNB.
#Drop-out mix:
Combine the appropriate ingredients, minus the relevant supplements, and mix in a sealed container. Turn the
container end-over-end for at least 15 minutes; add a few clean marbles to help mix the solids.
Adenine
0.5 g
Leucine
10.0 g
Alanine
2.0 g
Lysine
2.0 g
Arginine
2.0 g
Methionine
2.0 g
Asparagine
2.0 g
para-Aminobenzoic acid
0.2 g
Aspartic acid
2.0 g
Pheylalanine
2.0 g
Cysteine
2.0 g
Proline
2.0 g
Glutamine
2.0 g
Serine
2.0 g
Glutamic acid
2.0 g
Threonine
2.0 g
Glycine
2.0 g
Tryptophan
2.0 g
Histidine
2.0 g
Tyrosine
2.0 g
Inositol
2.0 g
Uracil
2.0 g
Isoleucine
2.0 g
Valine
2.0 g
1.5
TE buffer
Recipe
1.5.1 TE buffer, 10X
1.5.2 #100 mM Tris-Cl (desired pH)
1.5.3 10 mM EDTA (pH 8.0)
1.5.2 #100 mM Tris-Cl (desired pH):
1.5.2.1 Tris-Cl
1.5.2.1.1 Tris base
Tris base may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.
1.5.2.1.2 HCl
HCl (hydrochloric acid, hydrochloride) is volatile and may be fatal if inhaled, ingested, or absorbed through the
skin. It is extremely destructive to mucous membranes, upper respiratory tract, eyes, and skin. Wear appropriate
gloves and safety glasses. Use with great care in a chemical fume hood. Wear goggles when handling large
quantities.
To prepare a 1 M solution, dissolve 121.1 g of Tris base in 800 mL of H2O. Adjust the pH to the desired value
by adding concentrated HCl.
pH
HCl
7.4
70 mL
7.6
60 mL
8.0
42 mL
Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume
of the solution to 1 L with H2O. Dispense into aliquots and sterilize by autoclaving.
If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is
temperature-dependent and decreases ~0.03 pH units for each 1°C increase in temperature. For example, a 0.05 M
solution has pH values of 9.5, 8.9, and 8.6 at 5°C, 25°C, and 37°C, respectively.
1.5.3 10 mM EDTA (pH 8.0)
To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 mL of H2O. Stir vigorously
on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets). Dispense into aliquots and
sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is
adjusted to ~8.0 by the addition of NaOH.
Sterilize solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Store the buffer at room
temperature.
1.6 Yeast cultures
2. Equipment
2.1Camera
2.2Incubator preset to appropriate temperature for yeast culture
2.3Plates for yeast cultures (20)
2.4Spectrophotometer
2.5Tubes for liquid yeast cultures and for dilutions of yeast cultures
2.6Vortex mixer
METHOD
Preparation of 6AU Plates
1. Prepare a 50 mg/ml stock solution of 6AU in 1 M ammonium hydroxide. Prepare CSM/agar and cool
to ~55oC. To half of the CSM/agar, add 6AU to a final concentration of 100 µg/ml. Pour 10 CSM plates
and 10 CSM + 6AU plates.
Spotting
2. Grow 5 ml of yeast overnight at the appropriate temperature.
3. Vortex. Dilute a sample of yeast 1:20 in TE. Measure the OD600.
4. Dilute yeast to OD = 0.5 in TE (1 x 104 cells/µl). Label this Sample A.
For most strains, an OD600 of 0.5 is equivalent to ~1 x104 viable cells/µl. This will be different for
different strains and is dependent on plating efficiency.
5. Set up three tubes with 160 µl of TE and label as Samples B-D. Label an empty tube as Sample E.
6. Prepare a series of fivefold dilutions of yeast as follows:
i. Transfer 40 µl of A into B. Vortex.
ii. Transfer 40 µl of B into C. Vortex.
iii. Transfer 40 µl of C into D. Vortex.
iv. Transfer 40 µl of D into E.
7. Spot 5 µl of each sample on CSM and CSM + 6AU plates in duplicate. Grow at the appropriate
temperature. Photograph.