Freimoser:Fast yeast transformation protocol

Solutions/reagents: 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) watercells scrapped from plate</li>plate containing appropriate medium</li></ul> Equipment: Incubator</li>Centrifuge</li>Sterile 1.5-ml microcentrifuge tubes</li></ul> Steps: <ol> Measure out <font color=#357EC7>50 µl  of <a href="#carrier DNA" ><font color=#357EC7>carrier DNA </a> into sterile 1.5-ml microcentrifuge tube (1). </li> Add <font color=#357EC7>cells scrapped from plate. </li> Add <font color=#357EC7>1 - 2 µl  of <font color=#357EC7>DNA plasmid. Add <font color=#357EC7>240 µl  of <a href="#50% polyethylene glycol (PEG) solution" ><font color=#357EC7>50% polyethylene glycol (PEG) solution </a>. Add <font color=#357EC7>36 µl  of <a href="#1 M lithium acetate" ><font color=#357EC7>1 M lithium acetate </a>. </li> Mix solution by pipetting up and down several times. </li>  Option 1: Incubate  at <font color=#357EC7>30°C  for at least <font color=#357EC7>30 mins . (or) Option 2: Incubate at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for at least <font color=#357EC7>30 mins . </li>  Heat shock   Option 1: Store  at <font color=#357EC7>45°C  for <font color=#357EC7>15 mins . (or) Option 2: Store at <font color=#357EC7>42°C  for <font color=#357EC7>20 mins . </li> Centrifuge  at <font color=#357EC7>maximum speed for <font color=#357EC7>1 min  at <font color=#357EC7>room temperature , gently aspirate out the supernatant and discard it. Add <font color=#357EC7>100 µl  of <font color=#357EC7>water. Resuspend pellet by vortexing/by shaking vigorously. Plate out suspension onto <font color=#357EC7>plate containing appropriate medium. </li> </ol></ul></ul>