How to Perform a Yeast Transformation
Before you begin your transformation, first make sure you have enough of your materials, including: plasmid DNA, single stranded DNA 2, all buffers (0.1M LiOAc and freshly-made PEG3), and media (Yeast Extract-Peptone-Adenine-Dextrose liquid media...
Step-by-Step Guide
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Step 1: Before you begin your transformation
The liquid media and plates will take the longest to make if you are out. ,, Working close to the flame, transfer 5 milliliters (0.17 fl oz) of liquid Yeast Extract-Peptone-Adenine-Dextrosemedia to a culture tube with a serological pipet.
Scrape a small amount of cells off of the plate with a pipet tip, being careful not to puncture the solid media.
Carefully lower the tip into the tube, being careful not to touch the cells to the inner wall of the tube.
Pipet the liquid up and down until you can see the clump of cells on the tip dissolve.
Cover the tubes loosely to ensure oxygen flow to the cells.
Incubate the tubes in a 30⁰C shaker overnight.
Remove the cultures from the 30⁰C shaker from the night before and close the caps tightly.
Remove the plasmid DNA and single stranded DNA from the freezer (they are normally stored at
-20⁰C).
Leave the plasmid DNA to thaw at room temperature.
Heat the single stranded DNA in the 95⁰C hot plate.
The solution should become very hot (almost boiling) by the time single stranded DNA is used in the procedure. ,,,,, Tubes are normally labeled with the content (transformed plasmid), date, and scientist’s initials.,, Make sure to balance the centrifuge evenly.,,,,, PEG is a very viscous substance, so be careful when pipetting.
Pipet slowly, watching the buffer flow out of the tip.
If you pipet too quickly, there will still be PEG buffer left in the tube.,,,,,, Watch out for contamination at this step! Remove plates from the cold room, let warm to room temperature, and label each.
Use a sterilized stainless steel cell spreader, first dipped in ethanol, then passed through the flame to evaporate ethanol.
Let the spreader cool.
Touch a corner a corner of the spreader to the edge of the plate.
If the spreader makes an indentation in the media, the metal still too hot. ,,, Don’t leave too much of the plate open or contamination will occur more easily.,, Colonies should grow in 3-7 days. -
Step 2: first make sure you have enough of your materials
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Step 3: including: plasmid DNA
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Step 4: single stranded DNA 2
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Step 5: all buffers (0.1M LiOAc and freshly-made PEG3)
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Step 6: and media (Yeast Extract-Peptone-Adenine-Dextrose liquid media and plates with selective media).
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Step 7: The night (or late afternoon) before
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Step 8: make liquid cultures of the yeast cells you will be transforming.
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Step 9: You should also already have yeast cells growing on a plate with Yeast Extract-Peptone-Adenine-Dextrose or similar media.
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Step 10: Yeast Transformation: Now you should finally be ready to start the procedure.
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Step 11: Estimated time: minimum 2 hrs.
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Step 12: Set one hot plate to 95⁰C (can be between 90⁰ and 97⁰) and another to 30⁰C.
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Step 13: Turn on the Bunsen burner
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Step 14: and remember to work as close to the flame as possible.
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Step 15: Label clean Eppendorf tubes.
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Step 16: Pipet 600 uL of the liquid culture into each Eppendorf tube.
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Step 17: Centrifuge tubes for 2 min at 2000 rpm.
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Step 18: Remove supernatant by vacuum suction
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Step 19: being careful not to suck up any cells in the pellet.
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Step 20: Re-suspend pellet in 300 uL 0.1 M LiOAc buffer by pipetting or quickly vortexing the tubes.
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Step 21: Centrifuge again for 1 min at 2000 rpm and vacuum out supernatant.
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Step 22: Repeat steps 6 and 7 to make sure all media is washed out of the cells.
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Step 23: Pipet 600 uL of PEG buffer into each tube.
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Step 24: Pipet 5 uL of plasmid DNA and 10 uL of hot single stranded DNA into each tube.
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Step 25: Incubate the tubes on the 30ᵒ hot plate
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Step 26: anywhere from 30 minutes to overnight.
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Step 27: Heat shock the tubes in a 42ᵒ water bath for 15 min EXACTLY.
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Step 28: Centrifuge the tubes for 2 min at 3000 rpm and carefully vacuum out the supernatant.
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Step 29: Re-suspend the pellet in 100 uL MQ water.
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Step 30: Plate each sample on a separate selective plate.
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Step 31: Pipet the cell suspension from each Eppendorf tube onto the corresponding plate.
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Step 32: Spread the cells evenly with sterilized spreader by hand or on a rotating platform.
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Step 33: Dry plates around the flame with covers mostly on
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Step 34: leaving a small open space nearest to the flame.
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Step 35: As soon as they are dry
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Step 36: cover the plates and seal them with Parafilm.
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Step 37: Incubate the plates in a 30⁰ incubator.
Detailed Guide
The liquid media and plates will take the longest to make if you are out. ,, Working close to the flame, transfer 5 milliliters (0.17 fl oz) of liquid Yeast Extract-Peptone-Adenine-Dextrosemedia to a culture tube with a serological pipet.
Scrape a small amount of cells off of the plate with a pipet tip, being careful not to puncture the solid media.
Carefully lower the tip into the tube, being careful not to touch the cells to the inner wall of the tube.
Pipet the liquid up and down until you can see the clump of cells on the tip dissolve.
Cover the tubes loosely to ensure oxygen flow to the cells.
Incubate the tubes in a 30⁰C shaker overnight.
Remove the cultures from the 30⁰C shaker from the night before and close the caps tightly.
Remove the plasmid DNA and single stranded DNA from the freezer (they are normally stored at
-20⁰C).
Leave the plasmid DNA to thaw at room temperature.
Heat the single stranded DNA in the 95⁰C hot plate.
The solution should become very hot (almost boiling) by the time single stranded DNA is used in the procedure. ,,,,, Tubes are normally labeled with the content (transformed plasmid), date, and scientist’s initials.,, Make sure to balance the centrifuge evenly.,,,,, PEG is a very viscous substance, so be careful when pipetting.
Pipet slowly, watching the buffer flow out of the tip.
If you pipet too quickly, there will still be PEG buffer left in the tube.,,,,,, Watch out for contamination at this step! Remove plates from the cold room, let warm to room temperature, and label each.
Use a sterilized stainless steel cell spreader, first dipped in ethanol, then passed through the flame to evaporate ethanol.
Let the spreader cool.
Touch a corner a corner of the spreader to the edge of the plate.
If the spreader makes an indentation in the media, the metal still too hot. ,,, Don’t leave too much of the plate open or contamination will occur more easily.,, Colonies should grow in 3-7 days.
About the Author
Martha Gray
Experienced content creator specializing in cooking guides and tutorials.
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