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Yeast One Hybrid System
Yeast one hybrid system
I. Introduction II. Yeast Molecular Techniques III. Constructing a Reporter Vector for One-Hybrid
Analysis IV. Generating a cDNA Library V. Constructing & Screening a One-Hybrid Library VI. Analyzing Positive Interactions
Protein-DNA interaction
The differences between yeast one & two hybrid
II. Yeast Molecular Techniques
Yeast strains
A. Genotypes
II.Yeast Molecular Techniques
I. Introduction
I.INTRODUCTION
One-Hybrid System provides an in vivo genetic assay used for isolating novel genes encoding proteins that bind to a target, cis-acting regulatory element
D. Reporter genes
GAL1 UAS
GAL TATA
Lac Z
Yeast strains
II.Yeast Molecular Techniques
E. Leaky HIS3 expression
3-AT : 3-amino-1,2,4-triazole is a competitive inhibitor of the yeast HIS3 protein (His3p).3-AT is used to inhibit low levels of His3p expressed in a leaky manner and thus to suppress background growth on SD medium lacking histidine.
YEAST MEDIA
II.Yeast Molecular Techniques
A. YPD(YPDA) medium
20 g/L Difco peptone 10 g/L Yeast extract 20 g/L Agar (for plates only) (To 0.003% 0.2% adenine hemisulfate Solution) pH to 6.5 To 2% glucosesterile (40% stock solution)
YEAST MEDIA
II.Yeast Molecular Techniques
C.10X Dropout (DO) Solution
Nutrient
L-Adenine hemisulfate salt L-Arginine HCl L-Histidine HCl monohydrate L-Isoleucine L-Leucine L-Lysine HCl L-Methionine L-Phenylalanine L-Threonine L-Tryptophan L-Tyrosine L-Uracil L-Valine
Yeast can’t growth on selection medium
Yeast strains
II.Yeast Molecular Techniques
C. Mating type compatibilities
Y187 (MATα) can mate with AH109, HF7c, CG-1945, Y190, or SFY526 (all MATa).
Some yeast strains have relatively high basal levels of His3p.
Main difference between yeast and E.coli culture
Incubation time
Yeast
E.Coli
4~6 days
16 hours
B. Phenotypes
Yeast strain：Y187（MATα）
II.Yeast Molecular Techniques
Can’t synthesis His、 Leu、Trp by itself。
Incubate
Yeast
Yeast selection medium -His or -Leu or -Trp
ຫໍສະໝຸດ Baidu
Incubation temperature
Yeast
30℃
E.Coli
37℃
Culture medium
Yeast
E.Coli
YPD
LB
To optimize the 3-AT concentration in your selection medium:
Use the lowest concentration of 3-AT that, after one week, allows only small (<1 mm) colonies to grow. Too much 3-AT in the medium can kill freshly transformed cells.
YEAST MEDIA
II.Yeast Molecular Techniques
B.SD medium(1L)
6.7 g Yeast nitrogen base without amino acids 20 g Agar (for plates only) 850 ml H2O pH to 5.8 100 ml of the appropriate sterile 10X Dropout Solution 3-AT(1 M 3-AT stock solution) To 2% glucosesterile (40% stock solution)
Yeast one hybrid system
I. Introduction II. Yeast Molecular Techniques III. Constructing a Reporter Vector for One-Hybrid
Analysis IV. Generating a cDNA Library V. Constructing & Screening a One-Hybrid Library VI. Analyzing Positive Interactions
Protein-DNA interaction
The differences between yeast one & two hybrid
II. Yeast Molecular Techniques
Yeast strains
A. Genotypes
II.Yeast Molecular Techniques
I. Introduction
I.INTRODUCTION
One-Hybrid System provides an in vivo genetic assay used for isolating novel genes encoding proteins that bind to a target, cis-acting regulatory element
D. Reporter genes
GAL1 UAS
GAL TATA
Lac Z
Yeast strains
II.Yeast Molecular Techniques
E. Leaky HIS3 expression
3-AT : 3-amino-1,2,4-triazole is a competitive inhibitor of the yeast HIS3 protein (His3p).3-AT is used to inhibit low levels of His3p expressed in a leaky manner and thus to suppress background growth on SD medium lacking histidine.
YEAST MEDIA
II.Yeast Molecular Techniques
A. YPD(YPDA) medium
20 g/L Difco peptone 10 g/L Yeast extract 20 g/L Agar (for plates only) (To 0.003% 0.2% adenine hemisulfate Solution) pH to 6.5 To 2% glucosesterile (40% stock solution)
YEAST MEDIA
II.Yeast Molecular Techniques
C.10X Dropout (DO) Solution
Nutrient
L-Adenine hemisulfate salt L-Arginine HCl L-Histidine HCl monohydrate L-Isoleucine L-Leucine L-Lysine HCl L-Methionine L-Phenylalanine L-Threonine L-Tryptophan L-Tyrosine L-Uracil L-Valine
Yeast can’t growth on selection medium
Yeast strains
II.Yeast Molecular Techniques
C. Mating type compatibilities
Y187 (MATα) can mate with AH109, HF7c, CG-1945, Y190, or SFY526 (all MATa).
Some yeast strains have relatively high basal levels of His3p.
Main difference between yeast and E.coli culture
Incubation time
Yeast
E.Coli
4~6 days
16 hours
B. Phenotypes
Yeast strain：Y187（MATα）
II.Yeast Molecular Techniques
Can’t synthesis His、 Leu、Trp by itself。
Incubate
Yeast
Yeast selection medium -His or -Leu or -Trp
ຫໍສະໝຸດ Baidu
Incubation temperature
Yeast
30℃
E.Coli
37℃
Culture medium
Yeast
E.Coli
YPD
LB
To optimize the 3-AT concentration in your selection medium:
Use the lowest concentration of 3-AT that, after one week, allows only small (<1 mm) colonies to grow. Too much 3-AT in the medium can kill freshly transformed cells.
YEAST MEDIA
II.Yeast Molecular Techniques
B.SD medium(1L)
6.7 g Yeast nitrogen base without amino acids 20 g Agar (for plates only) 850 ml H2O pH to 5.8 100 ml of the appropriate sterile 10X Dropout Solution 3-AT(1 M 3-AT stock solution) To 2% glucosesterile (40% stock solution)