High Efficiency Transformation

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Day 2
Day 2
#Determine the titer of the yeast culture by pipetting 10 µl 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 106 cells/ml will give an OD600 of 0.1.  
#Determine the titer of the yeast culture by pipetting 10 µl 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 106 cells/ml will give an OD600 of 0.1.  
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#Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 108 cells to give 5 x 106 cells/ml.
#Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 108 cells to give 5 x 106 cells/ml.
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#Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
#Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
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#Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.
#Boil a 1.0 ml sample of carrier DNA for 5 min 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.
**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.
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#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.
#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.
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#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
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#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
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#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.
#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.
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#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
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#Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
#Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
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#Incubate the tubes in a 42°C water bath for 40 min.
#Incubate the tubes in a 42°C water bath for 40 min.
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#Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipettor.
#Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipettor.
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#Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
#Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
**We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants!!!!  
**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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#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 µl 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.
#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 µl 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.

Revision as of 11:27, 28 April 2005

High Efficiency Transformation Protocol


Day 1 Inoculate the yeast strain into 5 ml of liquid medium and incubate overnight.

Day 2

  1. Determine the titer of the yeast culture by pipetting 10 µl 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 106 cells/ml will give an OD600 of 0.1.
  2. Transfer 50 ml of YPD 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.
  4. Boil a 1.0 ml sample of carrier DNA for 5 min 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.
  1. 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.
  2. Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
  3. Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
  4. 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.
  5. 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) 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 Total 360 µl 2160 µl 3960 µl

  1. Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
  2. Incubate the tubes in a 42°C water bath for 40 min.
  3. Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipettor.
  4. Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
    • We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants!!!!
  1. 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 µl 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.

Notes:

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