Imran Mungrue imran.mungrue@utoronto.ca

RESUME

METHODS

7) Plasmid DNA Isolation

*Grow 1.5-2ml O/N bacterial culture with resistance antibiotic, depending on selectable marker of plasmid at 37degC. *Pellet at full speed in a microfuge, and aspirate supernatant. *Add 1.8ml Lysis Buffer to 0.2ml 10X Lysozyme/RNAse for every ten samples. 10X Lysozyme/RNAse: (Store @ -20degC) 5mg/ml Lysozyme 1mg/ml RNAse (Boiled) Lysis Buffer: (Store @ 4degC) 25ml  EDTA (500mM) 62.5   Triton X-100 (20%) 62.5   Sucrose (32%) 12.5   Tris-HCl pH8 (1M) 87.5   Water ------ 250 *Add (150) 200ul Lysis Buffer +Lysozyme/RNse to pelleted cells and vortex to mix. *Leave at RT for 10min. *Boil for 2min. *Cool on ice. *Spin @ full speed in a microfuge for 30min. *Toothpick out pelleted debris. *Add (150) 200ul isopropanol to remaining supernatant and vortex to mix. *Spin @ full speed for 10min in a microfuge. *Aspirate isopropanol and resuspend pellet in 50ul TE or water.