7. LB liquid medium: 10 g/L tryptone, 10 g/L NaCl, 5 g/L
yeast extract.
8. LB solid medium: 10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast
extract, 20 g/L agar.
9. 1000Â Zeocin stock solution: 100 mg/mL Zeocin
10. 1000Â Ampicillin stock solution: 100 mg/mL Ampicillin
11. DNA miniprep extraction kit.
12. BsrGI restriction enzyme with its reaction buffer.
2.4 Y2H Yeast
Strains
1. Y187: MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,
112, gal4Δ, met-, gal80Δ, MEL1, URA3:: GAL1UAS -GAL1TATA-lacZ.
2. AH109: MATa, trp1-901, leu2-3, 112, ura3-52, his3-200,
gal4Δ,
gal80Δ,
LYS2::
GAL1UAS-GAL1TATA-HIS3,
GAL2UAS-GAL2TATA-ADE2, URA3:: MEL1UAS-MEL1
TATA-lacZ.
2.5 Yeast
Culture Media
1. Yeast extract–peptone–dextrose (YPD) medium: 10 g/L yeast
extract 10 g/L, 20 g/L Bacto peptone 20 g/L, 20 g/L glucose, 100 mg/L adenine hemisulfate.
2. YPD solid medium: YPD medium plus 20 g/L Bacto agar.
3. Amino acid powder mix: Mix 6 g of each of the amino acids
listed in Table 1, supplement the mix with 6 g of adenine
hemisulfate.
4. 125Â supplement solutions: 20 mM uracil; 100 mM leucine;
100 mM histidine; 40 mM tryptophan. Histidine and tryptophan 125X solutions should be stored at 4
C and protected
from light
5. Synthetic Complete (SC) liquid Medium: 1.7 g/L yeast nitrogen base without amino acids, 5 g/L ammonium sulfate, 20 g/
L glucose, 1.3 g/L amino acid powder mix. Add 8 mL per liter
of the required 125Â supplement solutions to achieve the
desired selection condition as detailed in Table 2.
6. SC solid medium: SC medium plus 20 g/L Bacto agar.
Table 1
Amino acid list to supplement synthetic complete media
Alanine
Asparagine
Glutamine
Lysine
Proline
Tyrosine
Arginine
Cysteine
Glycine
Methionine
Serine
Valine
Aspartic acid
Glutamic acid
Isoleucine
Phenylalanine
Threonine
6
Monica Castro-Cruz et al.
yeast extract.
8. LB solid medium: 10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast
extract, 20 g/L agar.
9. 1000Â Zeocin stock solution: 100 mg/mL Zeocin
10. 1000Â Ampicillin stock solution: 100 mg/mL Ampicillin
11. DNA miniprep extraction kit.
12. BsrGI restriction enzyme with its reaction buffer.
2.4 Y2H Yeast
Strains
1. Y187: MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,
112, gal4Δ, met-, gal80Δ, MEL1, URA3:: GAL1UAS -GAL1TATA-lacZ.
2. AH109: MATa, trp1-901, leu2-3, 112, ura3-52, his3-200,
gal4Δ,
gal80Δ,
LYS2::
GAL1UAS-GAL1TATA-HIS3,
GAL2UAS-GAL2TATA-ADE2, URA3:: MEL1UAS-MEL1
TATA-lacZ.
2.5 Yeast
Culture Media
1. Yeast extract–peptone–dextrose (YPD) medium: 10 g/L yeast
extract 10 g/L, 20 g/L Bacto peptone 20 g/L, 20 g/L glucose, 100 mg/L adenine hemisulfate.
2. YPD solid medium: YPD medium plus 20 g/L Bacto agar.
3. Amino acid powder mix: Mix 6 g of each of the amino acids
listed in Table 1, supplement the mix with 6 g of adenine
hemisulfate.
4. 125Â supplement solutions: 20 mM uracil; 100 mM leucine;
100 mM histidine; 40 mM tryptophan. Histidine and tryptophan 125X solutions should be stored at 4
C and protected
from light
5. Synthetic Complete (SC) liquid Medium: 1.7 g/L yeast nitrogen base without amino acids, 5 g/L ammonium sulfate, 20 g/
L glucose, 1.3 g/L amino acid powder mix. Add 8 mL per liter
of the required 125Â supplement solutions to achieve the
desired selection condition as detailed in Table 2.
6. SC solid medium: SC medium plus 20 g/L Bacto agar.
Table 1
Amino acid list to supplement synthetic complete media
Alanine
Asparagine
Glutamine
Lysine
Proline
Tyrosine
Arginine
Cysteine
Glycine
Methionine
Serine
Valine
Aspartic acid
Glutamic acid
Isoleucine
Phenylalanine
Threonine
6
Monica Castro-Cruz et al.