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This protocol describes the production of competent yeast cells for lithium acetate/PEG DNA transformations.

1. In YPD, grow a 10 ml overnight yeast culture originating from a single colony. 2. Add the overnight culture to 190 ml of pre-warmed (30C) YPD and incubate at 30C until two doublings have occurred (see Hint #1). 3. Divide culture into four 50 ml sterile, disposable tubes and centrifuge 5 min at 1,000 X g. 4. Completely remove the supernatant, resuspend the pellets in 5 ml sterile ddH2O and combine the samples into two tubes. 5. Centrifuge the pooled samples for 5 min at 1,000 X g and remove the supernatant. 6. Resuspend the cells in 1 ml of TEL Buffer, combine the samples and transfer them to one 15 ml sterile disposable tube. 7. Incubate the cells for 1 hr at 30C using a roller drum to agitate the sample (see Hint #2). 8. The cells are now competent for transformation and may be used immediately or frozen (do not quick freeze) after adding sterile Glycerol to 15% (see Hint #3). Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version YPD Autoclave for 20 min 10 g/liter Yeast Extract 20 g/liter Glucose (Dextrose) 20 g/liter Peptone 1X TE Buffer 1X Lithium Acetate Buffer Prepare fresh with sterile ddH2O 1 M Lithium Acetate Autoclave or filter sterilize 10 mM EDTA Autoclave or filter sterilize 100 mM Tris, pH 7.5

TEL Buffer

Lithium Acetate (10X) TE Buffer (10X)

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable

Version Tris EDTA Glycerol Peptone Lithium Acetate Glucose Yeast Extract Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version 1. This will take about 4 hr. The final Optical Density at 600 nm (OD600) should be approximately 0.8. If strain is temperature sensitive, incubate at the permissive temperature. 2. If the strain used is temperature sensitive, incubate at the permissive temperature. 3. The efficiency of transformation will drop approximately 3 to 5 fold after freezing (see Protocol on Lithium Acetate Yeast Transformation).

FROZEN YEAST COMPETENT CELLS Making Frozen Yeast Competent Cells

1. Grow up intended stain (i.e. Y190) in appropriate media (YPAD) overnight. (Approximately 1ml for every 100ml intended to inoculate the next day (i.e. 2.5 ml overnight to inoculate 250ml). If you are growing a yeast strain with a selectable plasmid. Grow in appropriate selection media about 20 ml for every 100ml intended to inoculate the next day. Some yeast strains in selection media may take 2-3 days to reach saturation. You should grow just to the point of saturation.

2. Inoculate 250ml YPAD- this will be enough for about every seventy-five 100ul aliquots of frozen competent cells. Scale up as desired. Let them grow to log phase (~0.7 OD). Doubling time for Y190 is about 3.5 hours. If you are inoculating a strain with a selectable marker inoculate to ~OD 0.3 so it goes through one doubling time. You dont want too many doubling times as the may be lost due to non selection.

3. Spin down cells (6,000 for 10 minutes) and wash in 0.4 volumes of starting volume (i.e 40 ml if you grew up 100ml culture) of a 100mM solution of LioAC. (We do not

wash in water as in other protocols and this allows us to skip the 30 min incubation step)

4. Spin down cells again and wash in a 0.2 volumes starting volume with 100 mM LioAC.

5. Spin down a final time and resuspend cells in 100mM LioAC with 15% DMSO or glycerol to a final volume of 0.03 of your starting volume. Note: Glycerol seems to work better for us.

6. Aliquot 100ul shots in microfuge tubes and put cells into a cardboard box and allow to freeze slowly in 80C, unlike E. coli competent cells, flash freezing in liquid nitrogen will severely reduce their competency.

Solutions required: 1M Lithium Acetate (LioAC) 10.2 g in 100ml dH20 and autoclave. Dilute to 100mM with dH2O for working solution. Glycerol or DMSO

PREPARING COMPETENT YEAST CELLS FOR FROZEN STORAGE AND FOR TRANSFORMATION A. Making Competent Yeast Cells

1. Grow yeast cells in YPD (10 ml per transformation) to an OD600 of 0.6 to 1.0 from an inoculum of a single colony. This represents a cell density of approximately 0.6 to 1 X 107 cells/ml.

2. Centrifuge cells at 1,000 X g for 5 min in a table-top centrifuge to pellet the cells.

3. Resuspend the cells in 0.5 volume of Solution 1.

4. Centrifuge the cells at 1,000 X g for 5 min to pellet the cells.

5. Resuspend the cells in 0.02 volume of Solution 1 and split the solution into 0.2 ml aliquots.

6. Freeze the aliquots slowly (see Hint #2).

7. After freezing, store the aliquots at -70C until needed.

B. Transforming Yeast Cells

1. Add 0.1 to 5 g of plasmid DNA and 50 g of Carrier DNA in a maximum volume of 20 l on top of the frozen cell suspension.

2. Place the tube in a 37C water bath and mix every 10 to 15 sec until the solution begins to melt. Remove the tube from the water bath and warm it between gloved hands until melting is complete.

3. Add 1.4 ml of Solution 2 and mix by vortexing for 1 min.

4. Incubate at 30C for 1 hr (or permissive temperature if the strain is temperature sensitive).

5. Centrifuge cells at 5,000 rpm for 5 sec in a microcentrifuge.

6. Decant the supernatant and resuspend the pellet in 1 ml of Solution 3.

7. Plate an appropriate amount of cells onto selective media (SC plates).

8. Incubate cells at 30C (or permissive temperature, if using a temperature sensitive strain). Transformants should appear in 2 to 3 days (see Hint #3).

Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version 20% Glucose Autoclave 30 min SC Plates 900 ml ddH2O When plates are cool, add appropriate supplements. Pour plates 20 g Agar Add 100 ml 20% (w/v) Glucose Autoclave for 30 min Carrier DNA Solution 3 10 mg/ml Sonicated Salmon Sperm DNA 10 mM Bicine-NaOH, pH 8.3 6.7 g Yeast Nitrogen Base 20% (w/v) Glucose

Autoclave or filter sterilize

0.15 M NaCl Solution 2 40% (w/v) PEG 1000

0.2 M Bicine-NaOH, pH 8.3 Autoclave or filter sterilize Solution 1 1 M Sorbitol 5% (v/v) DMSO (CAUTION! see Hint #1) Autoclave or filter sterilize 3% (v/v) Ethylene Glycol YPD 10 g/liter Yeast Extract 10 mM Bicine-NaOH, pH 8.3

Autoclave 30 min 20 g/liter Glucose (Dextrose) 20 g/liter Peptone

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version Glucose Sorbitol DMSO Yeast Extract Yeast Nitrogen Base Peptone DNA, Salmon Sperm Ethylene Glycol Bicine Polyethylene Glycol (PEG) 1,000 Agar

Sodium Chloride Sodium Hydroxide

Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version 1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Slow freezing results in good viability.

3. Preparation of frozen competent cells from the two-hybrid strain PJ69-4A using the method above routinely gives results between 0.9 to 1.0 X 104 transformants/g DNA. This protocol has been reported to produce up to 105 transformants/g plasmid DNA.