Freimoser:Fast yeast transformation protocol

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Solutions/reagents:

• DNA plasmid
• carrier DNA
  
  (salmon/herring sperm DNA in H2O, 2mg/ml stock, 500 µl aliquots, heat-inactivated at 95°C for 5-6 min and put on ice before use (has to be done once))
• 50% polyethylene glycol (PEG) solution
  
  (MW 3'350, filter-sterilized)
• 1 M lithium acetate
  
  (autoclaved)
• water
• cells scrapped from plate
• plate containing appropriate medium

Equipment:

• Incubator
• Centrifuge
• Sterile 1.5-ml microcentrifuge tubes

Steps:

1. Measure out 50 µl of carrier DNA into sterile 1.5-ml microcentrifuge tube (1).
   
2. Add cells scrapped from plate.
   
3. Add 1 - 2 µl of DNA plasmid.
   Add 240 µl of 50% polyethylene glycol (PEG) solution.
   Add 36 µl of 1 M lithium acetate.
   
4. Mix solution by pipetting up and down several times.
   

5. Option 1: Incubate at 30°C for at least 30 mins.
   (or)
   Option 2: Incubate at room temperature for at least 30 mins.
   

6. Heat shock
   

   Option 1: Store at 45°C for 15 mins.
   (or)
   Option 2: Store at 42°C for 20 mins.
   

7. Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
   Add 100 µl of water.
   Resuspend pellet by vortexing/by shaking vigorously.
   Plate out suspension onto plate containing appropriate medium.