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:
- Measure out 50 µl of <a href="#carrier DNA" >carrier DNA</a> into sterile 1.5-ml microcentrifuge tube (1).
- Add cells scrapped from plate.
- 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>. - Mix solution by pipetting up and down several times.
Option 1: Incubate at 30°C for at least 30 mins.
(or)
Option 2: Incubate at room temperature for at least 30 mins.- Heat shock
Option 1: Store at 45°C for 15 mins.
(or)
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.
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