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Multiwell Transformation Protocol

Labor-saving implements

Multichannel pipettors--1 to 25 µl and 20 to 200 µl volume (Brinkmann 12-channel).
Sterile 96-well assay or tissue culture plate with lid (such as Falcon # 1172).
*Other sterile plates (flat, V, or U bottom) with well volumes of ~300 µl will also work.
Centrifuge for spinning multiwell plates.
Horizontal rotary shaker, to which a multiwell plate can be attached (by tape).
Sterile glass beads (5 mm diameter) + sterile spoon.
Solution troughs for multichannel pipettors (labcor #730-004).

Chemicals

PEG-3350 (Sigma P3640)
Lithium acetate dihydrate (Sigma L-6883)
Geneticin (Gibco BRL 11811-031)
10 mg/ml sheared salmon sperm DNA (Boehringer Mannheim 1467 140)
YPD broth
DMSO (Sigma )

Protocol

Two days beforehand:

1. Make YPD geneticin plates

Geneticin is dissolved in water at a concentration of 80 mg (dry weight) per ml and filter-sterilized.

Important!

The active concentration of Geneticin is written on the bottle and varies from lot to lot (from 500 to 800 µg per mg of total dry weight). The amount used per plate must be adjusted for each lot.

Gibco BRL reports that Geneticin is virtually indestructable (it can be autoclaved--though this has not been tried) and we have seen little decrease in strength after having plates sit at room temp for a month.

Geneticin is added to final active concentration of 300 µg/ml to 65 °C YPD broth containing 2 %Agar.

2. Dry plates for several days at room temperature.

(YPD is used instead of YPAD because we use a colony color assay to detect successful Ade2 control transformants.)

3. Start 2 ml liquid YPD (or YPAD) cultures of strains to be used in the transformation.

One day beforehad:

In the morning, dilute overnight yeast cultures 1:50 in 2 mls YPD (YPAD).

In the evening, (about 8 hours later) check O.D. 600 of the cultures. Hopefully the O.D.s should be between 1 and 5 (not in stationary phase).

Inoculate a flask containing 250 ml YPD (YPAD) with the appropriate volume of cells. This volume can be calculated using a simple formula. This volume will depend on the doubling time of the strain, and the number of hours before the transformation will be performed. The target O.D. 600 for the culture the next morning is about 1.5 - 2.0 . 250 ml is enough for 96 transformations, which we rarely do. More typically, we perform 36 haploid and 36 diploid transformations. In this case 100 ml of cells of each is enough.

On the day of the transformation:

1. Prepare 1 Liter sterile 100mM LiAcetate solution from sterile 1M stock solution.

2. Prepare Lithium Acetate PEG solution:

Mix exactly 15 gr PEG-3350 with 16.5 ml water (this is enough for 96 transformations). When PEG has dissolved, filter sterilize.

To 12 ml PEG solution
Add 1.5 ml sterile water
1.5 ml 1M LiAcetate

3. Turn on 30°C and 42°C water baths.

4. Boil carrier DNA for 10 minutes and then place on ice.

96-well transformation

1. Prepare cells

Check O.D. 600 of cells. The O.D. should be around 1.5 - 2.5. Cultures at lower densities give worse transformation efficiencies, even if you use twice as many cells (determined by Kexin Yu).

Pellet cells by centrifugation at room temperature. Dump off supernatant.

Wash cells 2 X in 0.5 vol 100 `mM LiAcetate

Resuspend cells in 1/100 vol 100 mM LiAcetate. If OD600 of culture was not 1.0 then adjust final volume. (If O.D.600 was 0.8, not 1.0, then resuspend 250 mls cells in 2.0 mls, not 2.5 ml etc.)

Place cells in a trough and add 1/9 vol carrier DNA (boiled for 10 minutes and then placed on ice.)

2. Place 5 µl PCR product into each well of a microtiter plate and add 25 µl of carrier DNA/cell mixture--mix well by pipetting up and down.

3. Incubate 15 minutes at 30°C.

4. Add 150 µl LiAcetate PEG solution--Mix well by pipetting up and down!

5. Incubate 30 minutes at 30°C (times of up to 1.5 hours have been used here).
6. Add 17 µl DMSO to each well.

7. Heat shock by placing the microtiter plate in a 42°C water bath for 15 minutes.

8. Pellet cells by centrifuging the microtiter plate 5 minutes in a low-speed centrifuge (we use a speed vac--no vacuum).

9. Remove PEG by tilting the plate and carefully drawing off PEG with a multichannel pipettor.
Try not to disturb the cell pellet, but some cell loss is inevitable.

10. Add 200 µl YPAD to each well and mix well.

11. Incubate microtiter plate at 30°C on a rotary shaker for ~4 hours~200 rpm.
The plate can be attached with tape to a rotary shaker in a warm room.

12. Plate entire contents of each well on a geneticin plate.

A row of plates (usually 12 at a time) is set up on the benchtop with lids ajar and some beads (20 or so) are spooned into each plate. The entire contents of a microtiter well is added to each plate. The lids are placed on the plates and the plates are stacked and the beads are swirled around to distribute the cells. Then the beads are dumped out. The beads are collected for re-use (following sterilization).

Using this method it takes us 3-4 hours to perform 96 transformations (one strain only) and about one and a half hours to plate out the cells. The procedure appears to work robustly in our hands and many variations, such as longer incubation times at the different steps) have been tried with little loss in efficiency. Please feel free to experiment yourself and let us know what you think works better.

Typical transformation results and interpretations.

Replica plating does not seem to be necessary if 300 µg/ml Geneticin is used. It is probably necessary if a lower concentration is used because the background increases.



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		Multiwell Transformation Protocol Labor-saving implements Multichannel pipettors--1 to 25 µl and 20 to 200 µl volume (Brinkmann 12-channel). Sterile 96-well assay or tissue culture plate with lid (such as Falcon # 1172). Other sterile plates (flat, V, or U bottom) with well volumes of ~300 µl will also work. Centrifuge for spinning multiwell plates. Horizontal rotary shaker, to which a multiwell plate can be attached (by tape). Sterile glass beads (5 mm diameter) + sterile spoon. Solution troughs for multichannel pipettors (labcor #730-004). Chemicals PEG-3350 (Sigma P3640) Lithium acetate dihydrate (Sigma L-6883) Geneticin (Gibco BRL 11811-031) 10 mg/ml sheared salmon sperm DNA (Boehringer Mannheim 1467 140) YPD broth DMSO (Sigma ) Protocol Two days beforehand: 1. Make YPD geneticin plates Geneticin is dissolved in water at a concentration of 80 mg (dry weight) per ml and filter-sterilized. Important!* The active concentration of Geneticin is written on the bottle and varies from lot to lot (from 500 to 800 µg per mg of total dry weight). The amount used per plate must be adjusted for each lot. Gibco BRL reports that Geneticin is virtually indestructable (it can be autoclaved--though this has not been tried) and we have seen little decrease in strength after having plates sit at room temp for a month. Geneticin is added to final active concentration of 300 µg/ml to 65 °C YPD broth containing 2 %Agar. 2. Dry plates for several days at room temperature. (YPD is used instead of YPAD because we use a colony color assay to detect successful Ade2 control transformants.) 3. Start 2 ml liquid YPD (or YPAD) cultures of strains to be used in the transformation. One day beforehad: In the morning, dilute overnight yeast cultures 1:50 in 2 mls YPD (YPAD). In the evening, (about 8 hours later) check O.D. 600 of the cultures. Hopefully the O.D.s should be between 1 and 5 (not in stationary phase). Inoculate a flask containing 250 ml YPD (YPAD) with the appropriate volume of cells. This volume can be calculated using a simple formula. This volume will depend on the doubling time of the strain, and the number of hours before the transformation will be performed. The target O.D. 600 for the culture the next morning is about 1.5 - 2.0 . 250 ml is enough for 96 transformations, which we rarely do. More typically, we perform 36 haploid and 36 diploid transformations. In this case 100 ml of cells of each is enough. On the day of the transformation: 1. Prepare 1 Liter sterile 100mM LiAcetate solution from sterile 1M stock solution. 2. Prepare Lithium Acetate PEG solution: Mix exactly 15 gr PEG-3350 with 16.5 ml water (this is enough for 96 transformations). When PEG has dissolved, filter sterilize. To 12 ml PEG solution Add 1.5 ml sterile water 1.5 ml 1M LiAcetate 3. Turn on 30°C and 42°C water baths. 4. Boil carrier DNA for 10 minutes and then place on ice. 96-well transformation 1. Prepare cells Check O.D. 600 of cells. The O.D. should be around 1.5 - 2.5. Cultures at lower densities give worse transformation efficiencies, even if you use twice as many cells (determined by Kexin Yu). Pellet cells by centrifugation at room temperature. Dump off supernatant. Wash cells 2 X in 0.5 vol 100 `mM LiAcetate Resuspend cells in 1/100 vol 100 mM LiAcetate. If OD600 of culture was not 1.0 then adjust final volume. (If O.D.600 was 0.8, not 1.0, then resuspend 250 mls cells in 2.0 mls, not 2.5 ml etc.) Place cells in a trough and add 1/9 vol carrier DNA (boiled for 10 minutes and then placed on ice.) 2. Place 5 µl PCR product into each well of a microtiter plate and add 25 µl of carrier DNA/cell mixture--mix well by pipetting up and down. 3. Incubate 15 minutes at 30°C. 4. Add 150 µl LiAcetate PEG solution--Mix well by pipetting up and down! 5. Incubate 30 minutes at 30°C (times of up to 1.5 hours have been used here). 6. Add 17 µl DMSO to each well. 7. Heat shock by placing the microtiter plate in a 42°C water bath for 15 minutes. 8. Pellet cells by centrifuging the microtiter plate 5 minutes in a low-speed centrifuge (we use a speed vac--no vacuum). 9. Remove PEG by tilting the plate and carefully drawing off PEG with a multichannel pipettor. Try not to disturb the cell pellet, but some cell loss is inevitable. 10. Add 200 µl YPAD to each well and mix well. 11. Incubate microtiter plate at 30°C on a rotary shaker for ~4 hours~200 rpm. The plate can be attached with tape to a rotary shaker in a warm room. 12. Plate entire contents of each well on a geneticin plate. A row of plates (usually 12 at a time) is set up on the benchtop with lids ajar and some beads (20 or so) are spooned into each plate. The entire contents of a microtiter well is added to each plate. The lids are placed on the plates and the plates are stacked and the beads are swirled around to distribute the cells. Then the beads are dumped out. The beads are collected for re-use (following sterilization). Using this method it takes us 3-4 hours to perform 96 transformations (one strain only) and about one and a half hours to plate out the cells. The procedure appears to work robustly in our hands and many variations, such as longer incubation times at the different steps) have been tried with little loss in efficiency. Please feel free to experiment yourself and let us know what you think works better. Typical transformation results and interpretations. Replica plating does not seem to be necessary if 300 µg/ml Geneticin is used. It is probably necessary if a lower concentration is used because the background increases.