High Efficiency Transformation

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High Efficiency Transformation Protocol
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{{back to protocols}}
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Day 0
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*Make sure you have the necessary solutions (instructions for how to make them can be found [http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/Solutions.html here]):
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*# Single-stranded carrier DNA
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*# PEG 3350 50% w/vol
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*# 1.0 M LiAc
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*Make sure you have enough plasmid DNA for your transformations. Each transformation should use between 0.5 and 1.0 µg of plasmid.
Day 1
Day 1
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Day 2
Day 2
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#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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#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 10<sup>6</sup> cells/ml will give an OD<sub>600</sub> 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.
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#Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 10<sup>8</sup> cells to give 5 x 10<sup>6</sup> cells/ml.
#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.
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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 (the next step).
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**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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#*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.
-
#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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#When the cell titer is at least 2 x 10<sup>7</sup> 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.
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#* If the cell titer is higher/lower than 2x10<sup>7</sup> cells/ml, you'll dilute it back down/up in a subsequent step
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#* Now would also be a good time to start warming up the 42°C water bath you'll need later on
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#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge at 3000 g for 30 sec and discard the supernatant.
#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.
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#* If your cell titer was higher/lower than 2x10<sup>7</sup> cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.
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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.
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#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at 3000g 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.  
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Number of Transformations
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:It's generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add 326 µl from the master mix to each transformation tube before adding the plasmid DNA + water component.
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Reagents 1 5 (6X) 10 (11X)
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:For the plasmid DNA + water component, you'll need to calculate, for each transformation, what vol. of your plasmid DNA solution contain the appropriate amount of plasmid DNA (0.5-1.0 µg). Add in the appropriate amount of plasmid DNA solution and then make it up to 34µl with water.
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PEG 3500 50% w/v 240 µl 1440 µl 2640 µl
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::{| border="1" cellpadding="2" cellspacing="0"
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LiAc 1.0 M 36 µl 216 µl 396 µl
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|-
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Boiled SS-carrier DNA 50 µl 300 µl 550 µl
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|
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Plasmid DNA plus Water 34 µl 204 µl 374 µl
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!colspan="3"|Number of Transformations
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Total 360 µl 2160 µl 3960 µl
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|-
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!Reagents||1||5 (6X)||10 (11X)
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|-
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|PEG 3350 50% w/v||240 µl||1440 µl||2640 µl
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|-
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|LiAc 1.0 M||36 µl||216 µl||396 µl
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|-
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|Boiled SS-carrier DNA||50 µl||300 µl||550 µl
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|-
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|Plasmid DNA plus Water||34 µl||204 µl||374 µl
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|-
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|'''Total'''||360 µl||2160 µl||3960 µl
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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.
#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.
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#Microcentrifuge at 3000 g for 30 sec and remove the Transformation Mix with a micropipettor.
#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 .
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**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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#*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.
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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 ml of water and plate 10 and 100 µl samples of the dilute 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:
Notes:
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*Based on Geitz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html
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*Based on Gietz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html
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*This protocol has been trimmed for efficiency. The original has some differences and some notes on how to achieve higher efficiency
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*See also the Gietz lab notes http://home.cc.umanitoba.ca/~gietz/
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*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency
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[[Category:Protocol]]
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[[Category:In vivo]]
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[[Category:Yeast]]

Current revision

back to protocols

Day 0

  • Make sure you have the necessary solutions (instructions for how to make them can be found here):
    1. Single-stranded carrier DNA
    2. PEG 3350 50% w/vol
    3. 1.0 M LiAc
  • Make sure you have enough plasmid DNA for your transformations. Each transformation should use between 0.5 and 1.0 µg of plasmid.

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 (the next step).
    • 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.
  5. 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.
    • If the cell titer is higher/lower than 2x107 cells/ml, you'll dilute it back down/up in a subsequent step
    • Now would also be a good time to start warming up the 42°C water bath you'll need later on
  6. Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge at 3000 g for 30 sec and discard the supernatant.
  7. Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
    • If your cell titer was higher/lower than 2x107 cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.
  8. Pipette 100 µl samples (ca. 108 cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at 3000g 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.
It's generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add 326 µl from the master mix to each transformation tube before adding the plasmid DNA + water component.
For the plasmid DNA + water component, you'll need to calculate, for each transformation, what vol. of your plasmid DNA solution contain the appropriate amount of plasmid DNA (0.5-1.0 µg). Add in the appropriate amount of plasmid DNA solution and then make it up to 34µl with water.
Number of Transformations
Reagents15 (6X)10 (11X)
PEG 3350 50% w/v240 µl1440 µl2640 µl
LiAc 1.0 M36 µl216 µl396 µl
Boiled SS-carrier DNA50 µl300 µl550 µl
Plasmid DNA plus Water34 µl204 µl374 µl
Total360 µl2160 µl3960 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 3000 g 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.
  5. 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 ml of water and plate 10 and 100 µl samples of the dilute 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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