Drummond:Transformation: Difference between revisions
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== Protocol == | == High-Efficiency Yeast Transformation Protocol == | ||
<br><br> ''Cell Competency Stage'' | |||
* | * Inoculate a 3 mL pre-culture of YPD with a single yeast colony and incubate overnight at 30⁰ C on a rotary wheel. | ||
* | * The next day, inoculate 50 mL of YPD (in a 500 mL baffled flask) with 500 µL of the overnight pre-culture (1:100 dilution). Check the OD600 to confirm that it is about 0.300 to 0.350. Incubate with shaking at 30⁰ C for 3.5 to 4 h at about 200 rpm. | ||
0. | |||
* | * Record the OD600 prior to harvesting. Pellet the cells at 3000 g for 2 min and wash once with 25 mL of sterile water. Pellet the cells at 3000 g for 2 min and resuspend in 1 mL of 100 mM LiOAc, and transfer to a sterile 1.5 mL Eppendorf tube.<br> Note: Make 100 mM LiOAc fresh from a 1 M stock solution. For 2 mL of 100 mM LiOAc, mix 200 µL of 1 M LiOAc with 1.8 mL of sterile water. | ||
* | * Pellet the cells at 20,000 g for 10 sec, remove the supernatant, and resuspend the pellet in 100 mM LiOAc to a final concentration of 2 X 109 cells/mL.<br> For example: If the OD600 = 1.667 (which is equivalent to 2 * 107 cells/mL), then <br>(50 mL) (2 * 107 cells/mL) = X (2 * 109 cells/mL) <br>X = 500 µL final volume of cells in 100 mM LiOAc | ||
* | * Incubate the cells at 30⁰ C in the bench top Thermomixer for 20 min at 1000 rpm. | ||
* | <br><br><br>''Transformation Stage'' | ||
* For each transformation, add 1 µg of transforming DNA and 10 µL of heat-denatured salmon sperm DNA (10 µg/µL) to a 50 µL aliquot of competent cells, and mix by vortexing.<br> Note: Heat denature salmon sperm DNA at 95⁰ C for 10 min, and immediately chill on ice for about 5 min before adding to an aliquot of competent cells | |||
* Add 300 µL of PEG/LiOAc solution, and mix by vortexing.<br>Note: Make the PEG/LiOAc solution fresh from 50% PEG3350, and 1 M LiOAc stock solutions. For 2 mL of PEG/LiOAc solution, mix 1.6 mL of PEG3350 with 200 µL of sterile water and 200 µL of 1 M LiOAc. | |||
* | * Heat-shock each transformation mixture at 42⁰ C for 40 min in a water bath. | ||
* | * Pellet each transformation mixture at 20,000 g for 10 sec, and wash once with 750 µL of sterile water. Pellet again, remove the wash and resuspend the cells in 200 µL of sterile water. Spread the cells onto appropriate selective media plates and incubate at 30⁰ C for 2-3 days. | ||
Latest revision as of 12:21, 25 November 2008
High-Efficiency Yeast Transformation Protocol
Cell Competency Stage
- Inoculate a 3 mL pre-culture of YPD with a single yeast colony and incubate overnight at 30⁰ C on a rotary wheel.
- The next day, inoculate 50 mL of YPD (in a 500 mL baffled flask) with 500 µL of the overnight pre-culture (1:100 dilution). Check the OD600 to confirm that it is about 0.300 to 0.350. Incubate with shaking at 30⁰ C for 3.5 to 4 h at about 200 rpm.
- Record the OD600 prior to harvesting. Pellet the cells at 3000 g for 2 min and wash once with 25 mL of sterile water. Pellet the cells at 3000 g for 2 min and resuspend in 1 mL of 100 mM LiOAc, and transfer to a sterile 1.5 mL Eppendorf tube.
Note: Make 100 mM LiOAc fresh from a 1 M stock solution. For 2 mL of 100 mM LiOAc, mix 200 µL of 1 M LiOAc with 1.8 mL of sterile water.
- Pellet the cells at 20,000 g for 10 sec, remove the supernatant, and resuspend the pellet in 100 mM LiOAc to a final concentration of 2 X 109 cells/mL.
For example: If the OD600 = 1.667 (which is equivalent to 2 * 107 cells/mL), then
(50 mL) (2 * 107 cells/mL) = X (2 * 109 cells/mL)
X = 500 µL final volume of cells in 100 mM LiOAc
- Incubate the cells at 30⁰ C in the bench top Thermomixer for 20 min at 1000 rpm.
Transformation Stage
- For each transformation, add 1 µg of transforming DNA and 10 µL of heat-denatured salmon sperm DNA (10 µg/µL) to a 50 µL aliquot of competent cells, and mix by vortexing.
Note: Heat denature salmon sperm DNA at 95⁰ C for 10 min, and immediately chill on ice for about 5 min before adding to an aliquot of competent cells - Add 300 µL of PEG/LiOAc solution, and mix by vortexing.
Note: Make the PEG/LiOAc solution fresh from 50% PEG3350, and 1 M LiOAc stock solutions. For 2 mL of PEG/LiOAc solution, mix 1.6 mL of PEG3350 with 200 µL of sterile water and 200 µL of 1 M LiOAc.
- Heat-shock each transformation mixture at 42⁰ C for 40 min in a water bath.
- Pellet each transformation mixture at 20,000 g for 10 sec, and wash once with 750 µL of sterile water. Pellet again, remove the wash and resuspend the cells in 200 µL of sterile water. Spread the cells onto appropriate selective media plates and incubate at 30⁰ C for 2-3 days.
