Freimoser:Fast yeast transformation protocol

<html><h2>Solutions/reagents:</h2><ul type="circle"><li>DNA plasmid</li><li> <a name="carrier DNA">carrier DNA <tab><div style="margin-right: 600px;">(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))</div></a></li><li> <a name="50% polyethylene glycol (PEG) solution">50% polyethylene glycol (PEG) solution <tab><div style="margin-right: 600px;">(MW 3'350, filter-sterilized)</div></a></li><li> <a name="1 M lithium acetate">1 M lithium acetate <tab><div style="margin-right: 600px;">(autoclaved)</div></a></li><li>water</li><li>cells scrapped from plate</li><li>plate containing appropriate medium</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><li>Measure out 50 µl of <a href="#carrier DNA" >carrier DNA</a> into sterile 1.5-ml microcentrifuge tube (1). </li><li>Add cells scrapped from plate. </li><li>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>. </li><li>Mix solution by pipetting up and down several times. </li><li>Option 1: Incubate at 30°C for at least 30 mins. (or) Option 2: Incubate at room temperature for at least 30 mins. </li><li>Heat shock Option 1: Store at 45°C for 15 mins. (or) Option 2: Store at 42°C for 20 mins. </li><li>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. </li></ol></ul></ul> </html>

Freimoser:Fast yeast transformation protocol

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