Plasmid Mini-Prep

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....

1. Pulse down cells in microcentrifuge on high (14,000 rpm) for 30 seconds.
2. Dump supernatent, suck off remaining culture, and resuspend cells in 100uL of ice cold Solution 1, vortexing ok.
3. Add 200uL of freshly made Solution 2. Rock gently, incubate on ice until clear (~ 5 minutes).
4. Add 150uL of ice cold Solution 3. Vortex immediately, incubate on ice ~ 5 minutes. For cleanest preps avoid transfering any precipitate.
5. Microcentrifuge on high (14,000 rpm) for 10 minutes, remove supernatant to new microcentrifuge tubes.
6. 10mM EDTA (pH 8.0)
7. Autoclave small batches of 100mL and store at 4 C.

   Solution 2

8. 0.1M NaOH (freshly diluted from 10M stock)
9. 1% SDS

   For 18 mini-preps (microcentrifuge head capacity)

10. 3.76 mL dH2O
11. 40uL 10M NaOH
12. 200uL 20% SDS
13. 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

14. 60mL 5M undefinedAcetate (Final Concentration 3M)
15. 11.5mL Glacial Acetic Acid (Final Concentration 5M)
16. 28.5mL dH2O