Freimoser:Fast yeast transformation protocol

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Solutions/reagents:

  • DNA plasmid
  • <a name="carrier DNA">carrier DNA
    <tab>
    (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))
    </a>
  • <a name="50% polyethylene glycol (PEG) solution">50% polyethylene glycol (PEG) solution
    <tab>
    (MW 3'350, filter-sterilized)
    </a>
  • <a name="1 M lithium acetate">1 M lithium acetate
    <tab>
    (autoclaved)
    </a>
  • 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 <a href="#carrier DNA" >carrier DNA</a> 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 <a href="#50% polyethylene glycol (PEG) solution" >50% polyethylene glycol (PEG) solution</a>.
    Add 36 µl of <a href="#1 M lithium acetate" >1 M lithium acetate</a>.
  4. Mix solution by pipetting up and down several times.
  5. <p>Option 1: Incubate at 30°C for at least 30 mins.
    (or)
    Option 2: Incubate at <b>room temperature</b> for at least 30 mins.

    </li>

  6. Heat shock
    <p>Option 1: Store at 45°C for 15 mins.
    (or)
    Option 2: Store at 42°C for 20 mins.

    </li>

  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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