OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM.SWP059
Initial Issue date
26.6.09
School/ Divisional Unit
School of Medical Science, Neuromuscular and
Regenerative Medicine Research Unit
Current version
Current Version
Next review date
1.0
26.6.12
Issue date
26.6.09
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
form.
Safe Work Procedure Title and basic description
Title:
Yeast 2-Hybrid analysis
Description: Testing of protein-protein interactions by expression of recombinant proteins in yeast.
Associated risk assessment title and location: 59_RA_Yeast 2-Hybrid_SP
Describe the activity or process
The design of a yeast 2-hybrid experiment may vary according to whether it concerns the
testing of a known interaction, screening a library or testing for auto-activation. However,
the essential elements involve transformation of yeast strains with plasmid DNA and the
plating of the transformed yeast on agar plates containing selective media.
This SWP is a generalized design that contains these elements. A real experiment will
require some thought with respect to negative and positive controls and which selective
media must be used.
Yeast transformation Geitz lab protocol
Day 1
Inoculate the yeast strain into 5 ml of liquid medium (2x YPD or SD selection medium) and incubate overnight on a
rotary shaker at 200 rpm and 30°C. Place a bottle of double strength YPD broth (2x YPD) and a 250 ml culture flask
in the incubator as well.
Day 2
1. Determine the titer of the yeast culture by pipetting 10 ml of cells into 1.0 ml of water in a spectrophotometer
cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10 6 cells/ml will give an
OD600 of 0.1. Alternatively, titer the culture using a hemocytometer.
i) Dilute overnight YPD or SD cultures 10-1 or more in water.
ii) Carefully place 10 µl of the cell suspension between the cover slip and the base of haemocytometer. Let the cells
settle onto the haemocytometer grid for a few minutes. The grid area is typically 1 square millimeter, divided into 25
equal-sized squares, and the volume measured is 10-4 ml.
iii) Count the number of cells in 5 diagonal squares
iv) Calculate the cell titer as follows: cells counted x 5 x dilution factor x 1/volume measured by the 25 squares of the
haemocytometer. 239 cells x 5 x 10 (dilution factor) x 1/10 -4ml = 1.2 x 108 cells/ml.
v) Saccharomyces cerevisiae divides by budding from a mother cell. Count budded cells as a single cells. Count cells
with equal bud sizes as two cells when there is evidence of additional buds forming on eithercell. Some strains form
clumps of cells which reduce plating efficiency. A single clump of cells will only give rise to one colony on a plate,
which may complicate further analysis.
2. Transfer 50 ml of the pre-warmed 2x YPD to the pre-warmed culture flask and add 2.5 x 108 cells to give 5 x 106
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cells/ml.
3. Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
Note:
i) It is important to allow the cells to complete at least two divisions.
ii)This will take 3 to 5 hours.
iii)This culture will give sufficient cells for 10 transformations.
iv) Transformation efficiency (transformants/ µg plasmid/10 8 cells) remains constant for 3 to 4 cell divisions.
4. When the cell titer is at least 2 x 107 cells/ml, which should take about 4 hours, harvest the cells by centrifugation at
3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.
5. Heat a 1.0 ml sample of salmon sperm carrier DNA for 5 min @ 100oC and chill in an ice/water bath while
harvesting the cells.
**** It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box
and boil after 3-4 freeze-thaws. But keep on ice when out.****
6. Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
7. Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
Note:If the cell titer of the culture is greater than 2 x 10 7 cells/ml the volume then increase the volume to maintain the
titer of this suspension at 2 x 109 cells/ml. If the titer of the culture is less than 2 x 10 7 cells/ml then decrease volume.
8. Pipette 100 µl samples (ca. 108 cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top
speed for 30 sec and remove the supernatant.
9. Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the
Transformation Mix in ice/water.
Number of Transformations
Reagents
1
5 (6X)
10 (11X)
240 µl
1440 µl
2640 µl
LiAc 1.0 M
36 µl
216 µl
396 µl
Boiled SS-carrier DNA
50 µl
300 µl
550 µl
Plasmid DNA plus Water
34 µl
204 µl
374 µl
360 µl
2160 µl
3960 µl
PEG 3500 50% w/v
Total
10. Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing
vigorously.
11. Incubate the tubes in a 42°C water bath for 40 min.
Note:The optimum time can vary for different yeast strains. Please test this if you need high efficiency from your
transformations.
12. Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipettor.
13. Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
Note:We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away
transformants!!!!
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14. Plate appropriate dilutions of the cell suspension onto SC selection medium. For transformation with an integrating
plasmid (YIp), linear construct or oligonucleotide, plate 200 µl onto each of 5 plates; for a YEp, YRp or YCp library
plasmid dilute 10 µl of the suspension into 1.0 ml of water and plate 10 and 100 µl samples onto two plates each. The
10 µl samples should be pipetted directly into 100 µl puddles of sterile water on the SC selection medium.
Note:When spreading yeast inoculum onto the plate gently distribute the fluid completely with a sterile glass rod with
a minimum of strokes. Allow the fluid to be taken up by the plate prior to incubation.
15. Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.
The transformation efficiency (transformants/1 µg plasmid/10 8 cells) can be determined by calculating the number of
transformants in 1.0 ml of resuspended cells per 1.0 microgram plasmid per 10 8 cells. For example, if the
transformation of 1.0 x 108 cells with 100 nanogram plasmid resulted in 500 colonies on a plate of SC dropout
medium spread with 1 µl of suspension (usually dispensed into a 100µl puddle of sterile water on the plate).
Transformation Efficiency = 500 x 1000 (plating factor) x 10 (plasmid factor) x 1 (cells/transformation x 10 8).
Transformation Efficiency = 5 x 106 transformants/1.0 µg plasmid/108 cells.
Transformation efficiency declines as plasmid concentration is increased (Gietz et al. 1995) but the actual yield of
transformants per transformation increases. For example, 100 nanogram of plasmid in a transformation might give a
Transformation Efficiency of 5 x 106 and a yield of 5 x 105 transformants whereas with 1 µg of plasmid the
Transformation Efficiency might be 2 x 106 and the yield 2 x 106 per transformation. In order to obtain 5 x 10 6
transformants it is simpler to set up two or three transformations with 1 µg of plasmid DNA, or a single 3 fold scaled
up transformation, than to carry out 10 reactions with 100 ng of plasmid in each.
Yeast Media and Plates
YPD medium
YPD Medium (Cat No. 630409) and YPD Agar Medium (Cat No. 630410) are available in powder
form from Clontech. Our YPD This is a blend of peptone, yeast extract, and dextrose in
optimal proportions for growth of most strains of Saccharomyces cerevisiae.
If you purchase Clontech’s YPD media, prepare the medium according
to the instructions provided. Alternatively, prepare your own YPD mixture as follows:
20 g/L Difco peptone
10 g/L Yeast extract
20 g/L Agar (for plates only)
• [Optional] For adenine-supplemented YPD (YPDA or YPAD), add 15 ml of a 0.2% adenine hemisulfate
solution per liter of medium (final concentration is 0.003%, in addition to the trace amount
of Ade that is naturally present in YPD). Adenine hemisulfate tolerates autoclaving.
Add H2O to 950 ml. Adjust the pH to 6.5 if necessary, then autoclave. Allow medium to cool
to ~ 55°C and then add dextrose (glucose) to 2% (50 ml of a sterile 40% stock solution). Adjust
the final volume to 1 L if necessary.
Note: If you add the sugar solution before autoclaving, autoclave at 121°C for 15 min; autoclaving at a higher
temperature,
for a longer period of time, or repeatedly may cause the sugar solution to darken and will decrease the performance
of the medium. Note that YPD from Clontech already contains glucose.
• [Optional] For kanamycin-containing medium, prepare YPD or YPDA as above. After autoclaved
medium has cooled to 55°C, add 0.2–0.3 ml of 50 mg/ml kanamycin (final concentration
10–15 mg/L).
SD medium
Minimal SD Base and Minimal SD Agar Base, either with dextrose (glucose), or galactose +
raffinose, are available from Clontech in powder form.
If you purchase Clontech’s Minimal SD Base, prepare your SD/Dropout (DO) medium according to
the instructions provided. For example, to prepare SD/–Leu/–Trp agar, you will need to combine
SD Minimal Agar Base (Cat No. 630412) with –Leu/–Trp DO Supplement (Cat No. 630412).
Alternatively, you can purchase yeast nitrogen base from another supplier (e.g., Difco Cat No.
0919-15-3) and prepare SD/DO medium as follows:
6.7 g Yeast nitrogen base without amino acids
20 g Agar (for plates only)
850 ml H2O
100 ml of the appropriate sterile 10X Dropout Solution
• Adjust the pH to 5.8 if necessary, and autoclave. Allow medium to cool to ~ 55°C before
adding 3-AT, cycloheximide, additional adenine, or X-gal (see below).
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• Add the appropriate sterile carbon source, usually dextrose (glucose) to 2%, unless specified
otherwise for your application. Adjust the final volume to 1 L if necessary.
Notes:
• If you add the sugar solution before autoclaving, autoclave at 121°C for 15 min; autoclaving at a higher temperature,
for a longer period of time, or repeatedly may cause the sugar solution to darken and will decrease the performance
of the medium. Note that SD Minimal Base from Clontech already contains a carbon source.
• If you purchase galactose separately, it must be highly purified and contain <0.01% glucose.
• [Optional] For 3-AT-containing medium, add the appropriate amount of 1 M 3-AT stock
solution and swirl to mix well. The concentration of 3-AT used in the medium depends on
the yeast strain and, to some extent, on the presence of transforming plasmid(s). See your
system-specific User Manual for further information.
Notes:
• 3-AT is heat-labile and will be destroyed if added to medium hotter than 55°C.
• 3-AT, a competitive inhibitor of the yeast HIS3 protein (His3p), is used to inhibit low levels
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Materials and Equipment:
 Benchtop centrifuge
 Incubator set at 30oC
 Spectrophotometer or Haemocytometer
 Petri dishes
 Conical flasks
 Shaking incubator set at 30oC
 Heating block set at set at 100oC
 Water bath or heating block set at 42oC
 Microcentrifuge
 Glass spreaders
Chemicals and solutions
 AH109 yeast cells

YPD and SD (with dropout supplements) Plates

YPD and SD (with DO) media

Sterile water

Polyethylene glycol (PEG) 3500 50% w/v

Lithium Acetate 1M

Salmon sperm DNA 10mg/ml

Plasmid DNA

3-amino-1,2,4-triazole

Betadine
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List potential hazards and risk controls including
specific precautions required
Mechanical/Electrical
Incubator set at 30oC - Electrical appliance – electrocution hazard
Spectrophotometer - Electrical appliance – electrocution hazard
Petri dishes
Conical flasks
Shaking incubator set at 30oC - Electrical appliance – electrocution hazard
Heating block set at set at 100oC - Electrical appliance – electrocution hazard –Burn hazard
Water bath or heating block set at 42oC - Electrical appliance – electrocution hazard
Microcentrifuge - Electrical appliance – electrocution hazard , spinning rotor- physical injury hazard.
Glass spreaders – Pasteur pipettes shaped in Bunsen flame – burn hazard, fragile glass, puncture wound hazard.
Chemical Hazards
Lithium Acetate 1M – Slightly hazardous in case of skin/eye contact (irritant) or inhalation/ingestion
3-amino-1,2,4-triazole - Slightly hazardous in case of skin/eye contact (irritant) or inhalation/ingestion
Risk controls
Standard PPE lab coat, goggles and gloves.
Electrical equipment testing
Irritant and toxic chemicals –weigh out and make solutions in fume hood
List emergency shutdown instructions
For electrical appliances, the emergency cutoff switch (red button) is located on the wall
near the entrance to the lab.
List clean up and waste disposal requirements
All solution s should be disposed of in chemical waste containers.
Solutions containing organic solvents i.e. phenol and chloroform should be put in a separate waste container to the aqueous waste
solutions .
Liquid waste contaminated with live yeast should be treated with Betadine solution before disposal in the biological waste container.
Contaminated solid waste should be disposed of through the biological and chemical waste stream.
List legislation, standards and codes of practice used
in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3:2002 Safety in laboratories Part 3: Microbiological aspects and containment facilities
Australian Standard AS2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
Australian Standard AS2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Safe Work Procedure Form (OHS026)
Supervisory approval, training, and review
Supervisor: Edna Hardeman
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction to Lab, Training in this SWP
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Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007