HIGH EFFICIENCY TRANSFORMATION

 

 

<center> <h2> <font>HIGH EFFICIENCY TRANSFORMATION</font></h2> </center>

 

<center> <h2> <font>Reference: Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.</font></h2> </center>

 

<center> <font><b><font>All Solutions for the LiAc/SS-DNA/PEG TRAFO Protocol are listed in the </font> <font>TRAFO Solutions Page.</font> </b> </font></center>

 

 

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High Efficiency Transformation Protocol

This protocol can be used to generate sufficient transformants in a single reaction to screen multiple yeast genome equivalents for plasmids that complement a specific mutation. It can also be used to transform integrating plasmids, DNA fragments and oligonucleotides for yeast genome manipulation. Finally, it is used to optimise the conditions for transformation of a particular yeast strain, for example, the transformation of a plasmid library into a two-hybrid yeast strain transformed with a bait plasmid by the Rapid Transformation Protocol. The High Efficiency Protocol can also be employed to transform a yeast strain simultaneously with two different plasmids, such as the two-hybrid bait and prey plasmids.

 

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Day 1

Inoculate the yeast strain into 5 ml of liquid medium (2x YPAD or SC selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30°C. Place a bottle of double strength YPAD broth (2x YPAD) and a 250 ml culture flask in the incubator as well.

 

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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. see note:

 

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i) Dilute overnight YPAD or SC cultures 10^-1 or more in water.

ii) Carefully place 10 µl of the cell suspension between the cover slip and the baseof 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.

ii) 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^-4 ml = 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.

 

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Note:

2. Transfer 50 ml of the pre-warmed 2x YPAD to the pre-warmed culture flask and add 2.5 x 10⁸ cells to give 5 x 10⁶ cells/ml.

3. Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.

Note:

• 1. 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/10⁸ cells) remains constant for 3 to 4 celldivisions.

 

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. Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.

⁸ 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	<center> <font><font><font><font><font><font><font><font><font><font><font><font><font><font>  <b>1</b> </font> </font> </font></font></font></font></font></font></font></font></font></font></font></font></center>	<center> <font><font><font><font><font><font><font><font><font><font><font><font><b><font>5 (6X)</font> </b> </font></font></font></font></font></font></font></font></font></font></font></font></center>	<center> <font><font><font><font><font><font><font><font><font><font><font><font><b><font>10 (11X)</font> </b> </font></font></font></font></font></font></font></font></font></font></font></font></center>
PEG 3500 50% w/v	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
<center> <font><font><font><font><font><font><font><font><font><font><font><font><b><font> Total</font> </b> </font></font></font></font></font></font></font></font></font></font></font></font></center>	360 µl	2160 µl	3960 µl

 

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.