酵母菌遺傳技術研習會
Yeast Genetic Skill Workshop
主辦：陽明大學基因體研究中心
酵母菌遺傳實驗室
1
目錄 :
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
1
Sporulation ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
6
Exp III. Isolation plasmid DNA from yeast ‧‧‧‧‧‧‧
7
Exp IV. Transformation ( Small scale ) ‧‧‧‧‧‧‧‧‧
9
Exp I.
Exp II.
Exp V.
Mating
Colony lift assay for β-galactosidase‧‧‧‧‧‧
Appendix A. Preparation of electrocompetent E. coli cells‧‧
13
14
Appendix B. Yeast two hybrid procedure‧‧‧‧‧‧‧‧‧ 15
2
Experiment I
:
Mating
<For small scale mating>
Day0.
Incubate bait strain and prey strain in 5 ml selection medium at
30℃ for 16-18 hr with shaking ( 200 rpm ). (Prepare by Yeast Genetic Lab)
Day1.
The OD600 value of both overnight cultures should be >0.8. Spin down the
cells by centrifugation at 1000 x g for 5 min. Resuspend cell pellets in 0.1
ml YPAD by vortexing. Combine the 0.1 ml PJ69-2A [bait] culture and the
0.1 ml Y187 [prey] culture in a tube. Then mix well by gentle inversion or
swirling. Drop 15~20 μl of the mixture onto 1 YPAD plates.
Incubate at 30℃ for 20-24 hr.
Day2
Add 5 ml sterile distilled water onto the plate and pipette up and down to
resuspend cells. Transfer the cell suspension into 50 ml cornical tubes.
Spin down the cell suspension by centrifugation at 1000 x g for 5 min
at 25℃. Wash the cell pellet with 10 ml sterile distilled water.
Centrifuge the cell suspension at 1000x g for 5 min at 25℃. Resuspend
the cell pellet in 0.2 ml distilled water. Spread 100μl of a 1:100 dilution of
the mating mixture on SD/－Leu, SD/－Trp, SD/－Leu/－Trp plates to
measure mating efficiency. Spread the remaining mating mixture on 1
selection plates. Incubate at 30℃ for 5-10 days.
Day7-14
Pick transformants onto selection plates.
3
Streak for single colonies.
Material :
Day0
5 ml SD/－Trp for PJ69-2A [bait]
5 ml
Day1
Day2
SD/－Leu for Y187 [prey]
1
YPAD plate
10 ml sterile distilled water
1
SD/－Leu plate
1
SD/－Trp plate
1
SD/－Leu/－Trp plate
1
selection plates
<Alternative experiments for library scale mating>
Method 1.
Day1.
Incubate bait strain in 100 ml selection medium at
30℃ for 16-18 hr with shaking ( 200 rpm ).
Day2.
The OD600 value of the overnight culture should be >0.8. Spin down the
cells ( about 100 OD unit ) by centrifugation at 1000 x g for 5 min.
Resuspend cell pellets in 1 ml YPAD by vortexing. Thaw out one frozen
aliquot (~1 ml) of the library culture in a 25℃ water bath. Gently vortex
the library aliquot. Combine the 1 ml PJ69-2A [bait] culture and the 1 ml
library culture in a tube. Then mix well by gentle inversion or swirling.
Drop 15~20 μl of the mixture onto 8 YPAD plates (about 12-15 drops/plate).
Incubate at 30℃ for 20-24 hr.
4
Day3
Add 5 ml sterile distilled water onto each plate and pipette up and down to
resuspend cells. Transfer the cell suspension into 50 ml cornical tubes.
Spin down the cell suspension by centrifugation at 1000 x g for 5 min
at 25℃. Wash the cell pellet with 50 ml sterile distilled water.
Centrifuge the cell suspension at 1000x g for 5 min at 25℃. Resuspend
the cell pellet in 2.5 ml distilled water. Spread 100μl of a 1:100, and
1:1000 dilution of the mating mixture on SD/－Leu, SD/－Trp,
SD/－Leu/－Trp plates to measure mating efficiency. Spread the remaining
mating mixture on 25 selection plates. Incubate at 30℃ for 5-10 days.
Day7-14
Pick transformants onto selection plates.
Streak for single colonies.
Material :
Day1
100 ml SD/－Trp for PJ69-2A [bait]
Day2
1 ml Gal4 system pretransformed library ( 109 cells/ml )
8
Day3
100 ml
2
2
2
25
YPAD plate
sterile distilled water
SD/－Leu plate
SD/－Trp plate
SD/－Leu/－Trp plate
selection plates
Method 2.
Day1.
Incubate bait strain ( PJ69-2A transformed with bait plasmid) in 100 ml
liquid SD/－Leu at 30℃ for 16-18 hr with shaking ( 200 rpm ).
Day2.
The OD600 value of the overnight culture should be >0.8. Spin down the
cells ( about 100 OD unit ) by centrifugation at 1000 x g for 5 min.
Resuspend cell pellets in the residual liquid (~5 ml) by vortexing. Thaw
out one frozen aliquot (~1 ml) of the library culture in a 25℃ water bath.
Gently vortex the library aliquot. Combine the entire PJ69-2A [bait]
culture and the library culture in a 2-L flask. Add 45 ml of YPAD/kan
and swirl gently. Use two 1 ml aliquots of YPAD/kan to rinse cells from
library tube. Incubate at 30℃ for 20-24 hr with gentle swirling (30-50
rpm).
5
Day3
Spin down the cells by centrifugation at 1000 x g for 10 min. Rinse the
mating flask two times with YPAD/kan (50 ml each rinse). Combine the
rinses and use them to resuspend the first pellet. Spin down the cells
again at 1000 x g for 10 min. Resuspend the cell pellet in 2.5 ml
distilled water. Spread 100μl of a 1:100, and 1:1000 dilution of the
mating mixture on SD/－Leu, SD/－Trp, SD/－Leu/－Trp plates
to measure mating efficiency. Spread the remaining mating
mixture on 25 selection plates. Incubate at 30℃ for 5-10 days.
Day7-14
Pick transformants onto selection plates.
Streak for single colonies.
Material :
Day1
100 ml SD/－Trp for PJ69-2A [bait]
Day2
1 ml Gal4 system pretransformed library (109 cells/ml)
50 ml
1
Day3
100 ml
2
2
YPAD/kan
2-L flask
YPAD/kan
SD/－Leu plate
SD/－Trp plate
2
SD/－Leu/－Trp plate
25
selection plates
Media and solutions :
 SD/－Trp :
1.7 g/L Bacto-yeast nitrogen base, 5 g/L ammonium sulfate,
0.7 g/L amino acid complex, 0.02 g/L Adenine, 0.02 g/L
Leucine, 0.02 g/L Histidine, 0.02 g/L Uracil, 0.02 g/L
Lycine, 2 % glucose
 YPAD/kan :
1 % Bacto-yeast extract, 2 % Bacto-peptone, 0.02g/L adenine,
2 % glucose, 50 mg/L kanamycin
 YPAD plate :
1 % Bacto-yeast extract, 2 % Bacto-peptone, 0.02g/L
adenine, 2 % glucose, 2 % agar
 SD/－Leu plate : 1.7 g/L Bacto-yeast nitrogen base, 5 g/L ammonium sulfate,
0.7 g/L amino acid complex, 0.02 g/L Adenine, 0.02 g/L
Tryptophan, 0.02 g/L Histidine, 0.02 g/L Uracil, 0.02 g/L
6
Lycine, 2 % glucose, 2 % agar
 SD/－Trp plate : 1.7 g/L Bacto-yeast nitrogen base, 5 g/L ammonium sulfate,
0.7 g/L amino acid complex, 0.02 g/L Adenine, 0.02 g/L
Leucine, 0.02 g/L Histidine, 0.02 g/L Uracil, 0.02 g/L
Lycine, 2 % glucose, 2 % agar
 SD/－Leu/－Trp plate : 1.7 g/L Bacto-yeast nitrogen base, 5 g/L ammonium
sulfate, 0.7 g/L amino acid complex, 0.02 g/L
Adenine, 0.02 g/L Histidine, 0.02 g/L Uracil, 0.02 g/L
Lycine, 2 % glucose, 2 % agar
 selection plate :
1.7 g/L Bacto-yeast nitrogen base, 5 g/L ammonium sulfate,
0.7 g/L amino acid complex, required amino acid 0.02 g/L,
2 % glucose, 2 % agar
7
Experiment II
:
Sporulation
Day1. Patch diploid yeast strain on YPAD plate. Incubate at 30℃ for 16-18 hr.
Day2. Replicate the YPAD plate to one sporulation plate (Alternatively, streak
diploid yeast on sporulation plate) .
Incubate at 25℃ for 2-4 days.
Day4-7. Check under microscope for the formation of tetrads.
Material :
Day1
Day2
1
1
1
1
YPAD plate
sporulation plate
replica set
sterile velvet
Media :

YPAD plate :

sporulation plate :
1 % Bacto-yeast extract, 2 % Bacto-peptone, 0.02g/L
adenine, 2 % glucose, 2 % agar
1 % potassium acetate, 0.02 g/L Adenine, 2 % agar
8
Experiment III
:
Isolation plasmid DNA from Yeast
(1) Yeast DNA preparation :
1.
Incubate yeast in 2 ml liquid medium at 30℃ for 16-18 hr with
2.
shaking( 200 rpm ).
Centrifuge 1.5 ml overnight culture at 13K rpm for 1 min at 25℃.
3.
4.
Decant the supernatant and resuspend the cell pellet in 0.1 ml STET solution.
Add ~0.2g 425-600 microns glass beads.
5.
6.
Vortex for 5 min.
Incubate at 100℃ for 3 min.
7.
8.
9.
Chill on ice for 5 min.
Centrifuge at 13K rpm for 10 min at 4℃.
Take 100μl supernatant to a new eppendorf. Add 50μl 7.5 M NH4OAc, mix well.
Incubate at -20℃ for 1 hr.
10. Centrifuge at 13K rpm for 10 min at 4℃.
11. Take 100μl supernatant to a new eppendorf. Add 100μl ice cold 100% EtOH,
mix well. Incubate at -70℃ for 10 min.
12. Centrifuge at 13K rpm for 10 min at 4℃.
13. Wash the pellet with 500μl 70% EtOH.
14. Dry the pellet, and then resuspend in 5μl sterile ddH2O.
Solution :
˙STET solution :
8 % sucrose, 50 mM Tris‧Cl
5 % Triton X 100
˙7.5M NH4OAc solution
9
pH 8.0, 50mM EDTA,
(2) Electroporation of E. coli
1.
2.
3.
4.
Chill sterile eppendorf tubes and electroporation cuvettes (1 mm or 2 mm gap) on
ice.
Set aside culture tubes (e.g. Falcon 2059 tubes) and SOC medium to culture the
bacterial cells after they have been electroporated.
Thaw the electrocompetent cells on ice. Aliquots of 40μl of cells are used for
standard electroporations.
Dispense aliquots of DNA into the prechilled eppendorf tubes. For example 2.6 μ
l of a solution of pUC19 DNA at a concentration of 100 pg/μl, can be used in
controls.
5.
Note that the DNA should be dissolved in sterile distilled water or in low
ionic strength buffer. Excessive salts can cause arcing.
Mix the DNA with the bacteria by pipetting up and down once or twice, and then
quickly transfer into the gap in the prechilled electroporation cuvette. Shack or tap
the cuvette so that the sample ends up at the bottom of the slot.
Keep the cuvette
8.
cold, and minimize the introduction of air bubbles.
Insert the loaded cuvette into the electroporator's Cuvette Holder. Close the Safety
Cover.
Look at the Pulse Indicator Light and press the Pulse Button. The Pulse Indicator
Light will flash and a tone will be emitted when a pulse is delivered.
Immediately add 1 ml of SOC to the cuvette to wash out the cells from the electrode
9.
gap, and transfer them into a culture tube.
Incubate at 30℃ for 1 hour with shaking (225 rpm).
6.
7.
10. Centrifuge cells at 14K rpm for 1 min.
11. Decant 900μl supernatant, and then resuspend cells in residual media.
12. Plate cells on LB agar plate containing the appropriate selective antibiotic.
Incubate the plate at 37℃ overnight.
Media and solutions :

SOC
:
2 % Bactotryptone, 0.5 % yeast extract, 10 mM NaCl, 2.5 mM KCl,
10 mM MgCl2, 10 mM MgSO4
－Prepare a 50X stock solution of the salts in distilled water.
－Prepare a 50X stock solution of glucose solution.
－Prepare the Bactotryptone/yeast extract solution.
Autoclave all three solutions separately and allow them to cool.
Add appropriate volume of the salts solution and glucose solution to
the Bactotryptone/yeast extract solution.
10
Experiment IV
:
Transformation ( Small scale )
Day 1.
Incubate PJ69-2A in 20 ml YPAD liquid at 30℃ for 16-18 hr with
shaking( 200 rpm ).
Day2.
Transfer overnight culture ( enough to produce an OD600 = 0.25- 0.3)
into 100 ml YPAD. Incubate at 30℃ for 3-5 hr with shaking( 200230 rpm). The OD600 will be 0.4- 0.6. Harvest cells by
centrifugation and follow the protocol for yeast transformation given
at the end of this section.
Day5-7.
Pick transformants onto selection plates.
Streak for single colonies.
Material :
Day1
20 ml liquid YPAD
Day2
100 ml liquid YPAD
50 ml sterile distilled water
50 ml sterile 1x TE/LiAc buffer ( 10 mM Tris, 1mM EDTA, pH 7.5 )
10 μg carrier DNA
0.1-1 μg plasmid DNA
1x PEG/TE/LiAc buffer (40% PEG4000 , 10 mM Tris, 1mM EDTA,
pH7.5)
1 selection plate
Media and solutions :
 YPAD :
1 % Bacto-yeast extract, 2 % Bacto-peptone, 0.02g/L adenine,
2 % glucose
 10x TE/LiAc buffer : 100 mM Tris, 10 mM EDTA, pH 7.5
 carrier DNA : 10 mg/ml autoclaved Calf thymus or herring sperm DNA, boil
fresh before use. (e.g. from Clontech or Promega )
 50 % (w/v) PEG4000 solution
 selection plate : 1.7 g/L Bacto-yeast nitrogen base, 5 g/L ammonium sulfate,
0.7 g/L amino acid complex, required amino acid 0.02 g/L,
2 % glucose, 2 % agar
11
TRANSFORMATION PROTOCOL
1. Place cells in 50 ml tubes and centrifuge at 1000 x g for 5 min at 25℃.
2. Discard the supernatant and resuspend cell pellet in 25 ml of 1x TE/LiAc buffer.
Mix cells by vortexing.
3. Centrifuge cell suspension at 1000x g for 5 min at 25℃.
4. Decant the supernatant and resuspend the cell pellet in 0.5 ml 1x TE/LiAc buffer.
5. Prepare PEG/TE/LiAc buffer 1 ml.
6. Mix 10μg carrier DNA and 0.1- 1μg plasmid DNA together with 100μl of cell
mixture from step 4 in a eppendorf tube.
7. Add 0.6 ml PEG/TE/LiAc buffer.
8. Vortex to thoroughly mix the sample.
9. Incubate at 30℃ for 30 min with shaking ( 200 rpm ).
10. Add 70μl of DMSO. Then mix well by gentle inversion or swirling. Do NOT
vortex.
11. Heat shock for 15 min in a 42℃ water bath. Swirl occasionally to mix.
12. Chill cells on ice for 5 min.
13. Centrifuge cells at 14K rpm for 5 sec at 25℃.
14. Decant the supernatant and resuspend cells in 0.1 ml of distilled water.
15. Plate cells on selection plate. Incubate at 30℃ for several days.
＜Alternative transformation procedure＞
(A)
Library scale transformation
1. Incubate PJ69-2A in 20 ml YPAD liquid at 30℃ for 16-18 hr with shaking(200
rpm).
2. Transfer overnight culture ( enough to produce an OD600 = 0.25- 0.3) into 300 ml
YPAD. Incubate at 30℃ for 3-5 hr with shaking( 200- 230 rpm). The OD600
will be 0.4- 0.6.
12
3. Place cells in 50 ml tubes and centrifuge at 1000 x g for 5 min at 25℃.
4. Discard the supernatant and resuspend cell pellet in 25 ml of 1x TE/LiAc buffer.
Mix cells by vortexing.
5. Centrifuge cell suspension at 1000x g for 5 min at 25℃.
6. Decant the supernatant and resuspend the cell pellet in 1.5 ml 1x TE/LiAc buffer.
7. Prepare PEG/TE/LiAc buffer 10 ml.
8. Mix 150-200μg carrier DNA and 10-50μg plasmid DNA together with 1 ml of
cell mixture from step 6 in a eppendorf tube.
9. Add 6 ml PEG/TE/LiAc buffer.
10. Vortex to thoroughly mix the sample.
11. Incubate at 30℃ for 30 min with shaking ( 200 rpm ).
12. Add 700μl of DMSO. Then mix well by gentle inversion or swirling. Do NOT
vortex.
13. Heat shock for 15 min in a 42℃ water bath. Swirl occasionally to mix.
14. Chill cells on ice for 5 min.
15. Centrifuge cells at 3K rpm for 5 min at 25℃.
16. Decant the supernatant and resuspend cells in 1 ml of 1x TE or sterile distilled
water.
17. Plate cells on selection plates. Incubate at 30℃ for several days
(B) 'Lazy Bones' transformation (plasmid transformations)
1. Take 0.5 ml of culture and spin 10 sec in microfuge. Decant the tube by inverting it
and shaking it once. Alternatively, one can pick a colony (2-3 mm in diameter)
from a plate with a toothpick and transfer cells to sterile 1.5 ml microfuge tube (as
long as the plate is not dried out, colonies can be used from plates stored in the
fridge for 3 months, maybe more).
2. Add 10 µl of carrier DNA (100 µg) plus 1 µg transforming DNA(in 10 µl) and
vortex well.
3. Add 0.5 ml PLATE solution and vortex.
4. Incubate overnight to 4 days at room temperature on the benchtop.
13
5. Heat shock for 15 min at 42 ℃.
6. Pellet cells in microfuge for a few seconds at 10 krpm. Carefully remove
supernatant.
7. Add 200 µl TE to the cell pellet and gently resuspend cells by aspirating
up-and-down with a pipette tip. Immediately spread suspended cells onto
selective plates.
NOTES
- the yield of transformants increases linearly up to about 100-200 µg of
transforming DNA.
- the optimal number of cells per transformation is about 2E8 cells/ml. Cells +
DNA volume should be about 140 µl. In other words, PLATE:(Cells + DNA)
should be about 3.5:1.
Solution:
˙PLATE solution :
40% PEG 3350, 0.1M LiAc, 10 mM Tris-HCl, pH 7.5,
1 mM EDTA
.
14
Experiment V
: Colony lift filter assay for β-galactosidase
1. Prepare Z buffer/X-gal solution.
2. Add 2 ml A buffer/X-gal solution to a clean 100-mm plate and layer a 75-mm
Whatman #1 filter onto the liquid to soak it.
3. Place a sterile Whatman #1 filter over the surface of the agar plate containing the
transformant colonies. To orient the filter on the agar, poke holes through the
filter into the agar, in three or more asymmetric locations.
4. Carefully lift the filter off the agar plate with forceps and transfer ( colonies
facing up ) to a pool of liquid nitrogen. Using the forceps completely submerge
the filter for 10 sec.
5. Remove the filter from liquid nitrogen and allow it to thaw at room temperature
buffer/ X-gal solution. Avoid trapping air bubbles under or between the filters.
Incubate the filters at 30℃ ( or room temperature) and check periodically for the
appearance of blue colonies.
Solutions :
‧ Z buffer
Na2HPO4˙7H2O 16.1 g/L, NaH2PO4H2O 5.5 g/L,
KCl
0.75 g/L,
MgSO4˙7H2O 0.246g/L, Verify that the pH is ~7.0; autoclave.
‧ X-gal stock solution
20 mg/ml X-gal in N,N-fimethylformamide (DMF). Store at -20℃.
‧ Z buffer/X-gal solution
10 ml
27 μl
167 μl
Z buffer
β-mercaptoethanol
X-gal stock solution
Note: Colony lift filter assay can be performed at any time after colonies are visible.
However, the best results will be obtained using freshly transformed colonies,
1-2 mm in diameter.
15
Appendix A :
Preparation of electrocompetent E. coli cells
1.
Inoculate a culture flask containing 50 ml SOB from a bacterial colony on the LB
plate. Incubate at 37℃ overnight with shaking (130 rpm).
2.
Inoculate a 2-L flask containing 500 ml SOB with 5 ml of the overnight culture.
Incubate at 37℃ with shaking (180 rpm) until the cell density reaches an optical
3.
4.
5.
density of 0.8 at 550 nm (2-4 hr).
Chill the culture flask and two 250 ml centrifuge bottles on ice.
The cells have to
be kept ice-cold throughout the following steps if high transformation efficiencies
are to be attained.
Transfer the culture into the chilled centrifuge bottles and collect the cells by
centrifugation(15 min at 2500 x g at 4℃) .
Decant the supernatant.
Wash the cells by resuspending them in ice-cold sterile
distilled water. Use about 200 ml of water to resuspend both pellets and transfer all
the cells into one bottle. Centrifuge at 2500 x g for 15 min at 4℃).
6.
7.
8.
9.
Repeat step 5.
Wash the cells twice in ice-cold sterile 10 % glycerol. Take care not to dislodge too
many cells from the pellet while decanting the supernatant.
A small volume of ice-cold sterile glycerol may have to be added to the cell pellet to
resuspend the cells at a density 100-200 OD500 units. An OD500 of 10 corresponds
to 4-8 X 109 bacteria/ml. Higher cell concentrations yield higher transformation
efficiencies.
Dispense aliquots of the cell suspension in prechilled eppendorf tubes. Store at -70
℃.
Media and solutions :

SOB
:
2 % Bactotryptone, 0.5 % yeast extract, 10 mM NaCl, 2.5 mM KCl,
10 mM MgCl2, 10 mM MgSO4
－Prepare a 50X stock solution of the salts in distilled water.
－Prepare the Bactotryptone/yeast extract solution.
Autoclave all two solutions separately and allow them to cool.
Add appropriate volume of the salts solution to the
Bactotryptone/yeast extract solution.

Sterile 10 % glycerol solution in distilled water
16
Appendix B :
Yeast two hybrid procedure
Step 1. Construct Gal4 DNA-BD/target plasmid (bait).
Step 2. Transform PJ69-2A with bait plasmid ( Exp. 4 ) and test for autonomous
reporter gene activation and cell toxicity.
Step 3. Mate the pretransformed bait and library cultures ( Exp. 1 ).
Step 4. Streak transformants that can grow on selection media.
Step 5. Perform β-gal colony -lift filter assay (Exp. 5).
Step 6.
Step 7.
Isolation plasmid DNA from positive yeast clones (Exp. 3).
Identify and isolate AD/library plasmids from E. coli transformants.
Step 8. Retransform plasmid into Y187 and mate with PJ69-2A [bait] or PJ69-2A
[pAS2-1] .
Step 9. Repeat Step 4 & 5.
Step 10. Select true positive clones and check their library DNA sequence.
17