Objective:

Mini-Prep procedure is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis, sequencing, cloning, or other purposes, but should not be used with out additional cleanup for embryonic injections.

Procedures:

Start => Plasmid containing bacterial hosts in liquid culture....

Pulse down cells in microcentrifuge on high (14,000 rpm) for 30 seconds. Dump supernatent, suck off remaining culture, and resuspend cells in 100uL of ice cold Solution 1, vortexing ok. Add 200uL of freshly made Solution 2. Rock gently, incubate on ice until clear (~ 5 minutes). Add 150uL of ice cold Solution 3. Vortex immediately, incubate on ice ~ 5 minutes. For cleanest preps avoid transfering any precipitate. Microcentrifuge on high (14,000 rpm) for 10 minutes, remove supernatant to new microcentrifuge tubes. ** For cleanest preps avoid transfering any precipitate. Add 1mL of isopropanol to each tube (fill microcentrifuge tube). Cold isopropanol or incubation on ice may increase DNA precipitation yields. ** May be stored in freezer at this stage if necessary. Microcentrifuge on high (14,000 rpm) for 10 minutes, carefully discard supernatant, and add 1mL of 70% EtOH to wash pellet by gently mixing. ** Recentrifuge if pellet is disturbed for 5-10 min on high. Remove all supernatent with a pulled pasteur pippette, and allow remnants to evaporate on bench or in dessicator until pellet is just dry. ** Problems may be encountered drying to resuspend the pellet if allowed to overdry. Add 2uL of RNase A (10ug/mL) to 1 mL of TE buffer. Add 50uL of RNase A TE to pellet to resuspend, and incubate at 37 degrees for ~20 minutes.

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Reagents/Solutions Solution 1

• 50mM glucose
• 25mM Tris*Cl (pH 8.0)
• 10mM EDTA (pH 8.0)
• Autoclave small batches of 100mL and store at 4 C. Solution 2
• 0.1M NaOH (freshly diluted from 10M stock)
• 1% SDS For 18 mini-preps (microcentrifuge head capacity)
• 3.76 mL dH2O
• 40uL 10M NaOH
• 200uL 20% SDS
• Sambrook et al. calls for 0.2M NaOH; however, SCF believes that more nicking may occur at the higher concentration and there is no significant loss of yield. Solution 3
• 60mL 5M K*Acetate (Final Concentration 3M)
• 11.5mL Glacial Acetic Acid (Final Concentration 5M)
• 28.5mL dH2O