Team:Gaston Day School/22 June 2009

 High eff. EcoRI: RFP, constit. RFP Thaw e. Coli till ice disappears Pipette 100x into tube (X2) Add 10x of plasmid to e. Coli Place on ice for 30 min Heat shock at 42C for 30 sec Ice for 5 mins Add 950x SOC</dt> Place at 37C for 60 mins</dt> Spin 5 mins</dt> Pipette out SOC</dt> Resuspend in 150x SOC</dt> Spread 50x and 100x onto amp plates</dt> More mini prep</dt> Spin tubes #1-8 for 3 minutes</dt> Combine tubes into #8</dt> Add 300x TENS</dt> Mix by inversion</dt> Add 150x sodium acetate</dt> Mix by inversion</dt> Spin 2.5 mins</dt> Transferred Sup to clean tube added 1mL of EtOH (Ethanol)</dt> Spin for 5 mins</dt> Washed pellet in 70% EtOH 300x</dt> <dt>Spin 5 mins</dt> <dt>pst = B#3</dt> <dt>Spe and Xba = B#4</dt> <dt>Resuspend 30x TE</dt> <dt>Pst = B #3</dt> <dt>Spe and ba = B#4</dt> <dt>DIGEST</dt> <dt>10x DNA</dt> <dt>3x Buffer/B#3/B#4</dt> <dt>15x dH2O</dt> <dt>2x Eco RI/Pst/SPe/Xba</dt>