Freimoser:Fast yeast transformation protocol

From OpenWetWare
Jump to: navigation, search

<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 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></i></a></li><li> <a name="50% polyethylene glycol (PEG) solution">50% polyethylene glycol (PEG) solution <i><br><tab><div style="margin-right: 600px;">(MW 3'350, filter-sterilized)</div></i></a></li><li> <a name="1 M lithium acetate">1 M lithium acetate <i><br><tab><div style="margin-right: 600px;">(autoclaved)</div></i></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><p><li>Measure out <b><font color=#357EC7>50 µl</font></b> of <a href="#carrier DNA" ><font color=#357EC7>carrier DNA</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br></li></p><p><li>Add <font color=#357EC7>cells scrapped from plate</font>.<br></li></p><p><li>Add <b><font color=#357EC7>1 - 2 µl</font></b> of <font color=#357EC7>DNA plasmid</font>.<br>Add <b><font color=#357EC7>240 µl</font></b> of <a href="#50% polyethylene glycol (PEG) solution" ><font color=#357EC7>50% polyethylene glycol (PEG) solution</font></a>.<br>Add <b><font color=#357EC7>36 µl</font></b> of <a href="#1 M lithium acetate" ><font color=#357EC7>1 M lithium acetate</font></a>.<br></li></p><p><li>Mix solution by pipetting up and down several times.<br></li></p><p><li><p><b>Option 1: </b>Incubate at <b><font color=#357EC7>30°C</font></b> for at least <b><font color=#357EC7>30 mins</font></b>.<br>(or)<br><b>Option 2: </b>Incubate at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for at least <b><font color=#357EC7>30 mins</font></b>.<br></p><p></li></p><p><li><b><font size=3>Heat shock</font></b><br><p><b>Option 1: </b>Store at <b><font color=#357EC7>45°C</font></b> for <b><font color=#357EC7>15 mins</font></b>.<br>(or)<br><b>Option 2: </b>Store at <b><font color=#357EC7>42°C</font></b> for <b><font color=#357EC7>20 mins</font></b>.<br></p><p></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>1 min</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>100 µl</font></b> of <font color=#357EC7>water</font>.<br>Resuspend pellet by vortexing/by shaking vigorously.<br>Plate out suspension onto <font color=#357EC7>plate containing appropriate medium</font>.<br></li></p></ol></ul></ul> </html>