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Turbo Plasmid DNA Midi-Prep 1. Inoculate 50 ml LB + antibiotic with one fresh colony of E. coli + plasmid. Grow overnight at
 

Summary: Turbo Plasmid DNA Midi-Prep
1. Inoculate 50 ml LB + antibiotic with one fresh colony of E. coli + plasmid. Grow overnight at
37C (12 18 hours). Next morning, transfer to 50 ml tube and chill on ice.
2. Centrifuge for 30 minutes at ~1000 g at 4C. Remove supernatant. Pellet may be frozen.
3. Resuspend pellet in 50 ml tube with 1 ml TE or GTE buffer.
4. Add 4 ml of freshly made 0.2 N NaOH + 1% SDS. Invert tube gently until all clumps are
dispersed. Do not vortex. Place on ice 5 minutes.
5. Add 3 ml of 3M KOAc, pH 5. Shake gently to disperse clumps. Place on ice for 5 minutes.
6. Add 2 ml CHCl3:isoamyl alcohol (24:1). Shake gently to mix.
7. Centrifuge for 10 minutes at ~1000 g at 4C.
8. Transfer supernatant (~7.5 ml) to 50 ml tube (use pipette and measure volume).
9. Add 2 volumes of 100% ethanol. Invert to mix. Chill on ice 10 minutes.
10. Centrifuge for 10 minutes at ~1000 g at 4C. Pour off supernatant and drain tube on Kimwipe.
11. Add 1.0 ml of 70% ethanol, repipette to disperse pellet, and transfer to a microfuge tube.
12. Microfuge for 1 minute at top speed. Remove as much supernatant as possible.
13. Repeat 70% EtOH wash (steps 11 and 12).
14. Add 500 l of TE. Vortex/repipette until pellet is completely dissolved.
15. Add 1 l of heat-treated 10 mg/ml RNase A. Incubate at 37C for 30 - 60 minutes. Place on ice.
16. Microfuge at top speed for 10 minutes. Transfer supernatant to a fresh tube and measure volume.
17. Add an equal volume of 1.6 M NaCl + 13% PEG 8000. Mix completely (PEG solution is

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine