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Procedures:

Start = 2mL bacterial cultures containing plasmids of interest that have grown 12-18 hours at 37.

   1. Pour ~1.4 mL of the culture into labeled 1.5 mL microcentrifuge tubes. Place the borosilicate glass tubes with the remaining cultures at 4C; they can be used to start additional culture for several weeks.
   2. Spin down cells in microcentrifuge on high (14,000 rpm) for 1 min.
   3. Aspirate out supernatant and resuspend cells in 250L of Solution 1. It is important to resuspend all the cells and not leave large pellet of cells in the tube.
   4. Add 300L of Solution 2. Mix by gently inverting and rolling the tube to make sure all the bacterial slurry along the sides and cap are mixed with solution 2,
   5. Add 350L of Solution 3. Gently mix by inverting the tube.
   6. Microcentrifuge at 12,000-14,000 x g for 10 minutes, pour supernatant to new microcentrifuge tubes. ** For cleanest preps avoid transferring any precipitate.
   7. Add 0.6mL of isopropanol to each tube (fill microcentrifuge tube w/ squirt bottle). Mix by inverting several times.
   8. Microcentrifuge at 12,000-14,000 x g for 10 minutes, carefully discard supernatant, and add ~1mL of 70% EtOH with a squirt bottle to wash pellet and gently mix by inverting. ** If pellet is disturbed recentrifuge for 5 minutes at 12,000-14,000 x g.
   9. Pour supernatant into ethanol waste container, remove remaining EtOH with a pipettor, and allow remnants to evaporate on bench or in dessicator until pellet is just dry. ** Problems may be encountered trying to resuspend the pellet if allowed to overdry.
  10. Add 2L of RNase A (10ug/mL) to 1 mL of TE buffer. Add 50L of RNase A TE to pellet to resuspend, and incubate at 37 for ~20 minutes.

Reagents/Solutions
Solution 1

    * 50mM glucose
    * 25mM Tris*Cl (pH 8.0)
    * 10mM EDTA (pH 8.0)
     
Solution 2

    * 0.1M NaOH (freshly diluted from 10M stock)
    * 1% SDS

Solution 3

    * 60mL 5M K*Acetate (Final Concentration 3M)
    * 11.5mL Glacial Acetic Acid (Final Concentration 5M)
    * 28.5mL dH2O