Freimoser:Fast yeast transformation protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>DNA plasmid</li><li> <a name="carrier DNA">carrier DNA <i><br><tab><div style="margin-right: 600px;">(salmon/herring sperm DNA in H2...)
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Current revision

Solutions/reagents:

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.

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