Freimoser:Fast yeast transformation protocol
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Vaishnavi Ananth (Talk | contribs)
(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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- 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)
- cells scrapped from plate
- plate containing appropriate medium
- Sterile 1.5-ml microcentrifuge tubes
- Measure out 50 µl of carrier DNA into sterile 1.5-ml microcentrifuge tube (1).
- Add cells scrapped from plate.
- Add 1 - 2 µl of DNA plasmid.
Add 240 µl of 50% polyethylene glycol (PEG) solution.
Add 36 µl of 1 M lithium acetate.
- Mix solution by pipetting up and down several times.
Option 1: Incubate at 30°C for at least 30 mins.
Option 2: Incubate at room temperature for at least 30 mins.
- Heat shock
Option 1: Store at 45°C for 15 mins.
Option 2: Store at 42°C for 20 mins.
- 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.