Home

About

Advanced Search

Browse by Discipline

Scientific Societies

E-print Alerts

Add E-prints

E-print Network
FAQHELPSITE MAPCONTACT US


  Advanced Search  

 
Plasmid Maxi-Prep On CsCl Gradients 1. Grow 1 liter of E. coli + plasmid overnight at 37C in LB + antibiotic. Transfer culture volume to
 

Summary: 22
Plasmid Maxi-Prep On CsCl Gradients
1. Grow 1 liter of E. coli + plasmid overnight at 37C in LB + antibiotic. Transfer culture volume to
four 250 ml centrifuge bottles. Chill cells on ice for 10 minutes.
2. Centrifuge at 4C for 10 minutes at 5500 rpm (~5000 g) in Sorvall GSA rotor.
3. Remove as much supernatant as possible. Add 40 mls of GTE and resuspend pellet completely by
repipetting. Add 50 mg Lysozyme and swirl to dissolve. Hold at room temperature for 10 minutes.
4. Add 80 mls of freshly made 0.2 N NaOH + 1% SDS (dilute 2 M NaOH and 10% SDS). Mix by
swirling until all clumps are dispersed. Place on ice 15 minutes.
5. Add 60 ml of 3M KOAc, pH 5. Swirl liquid in centrifuge bottle while adding KOAc slowly. Mix
completely. Stir gently with glass rod if clumps don't disperse. Place on ice 15 minutes.
6. Centrifuge at 4C for 10 minutes at 5500 rpm in Sorvall GSA rotor.
7. Pour supernatant into 250 ml bottle through 4 layers of cheesecloth to remove small pieces of
precipitate. Total volume should be about 150 mls (estimate total volume by eye).
8. Add 90 mls (0.6 volumes) of isopropanol. Mix. Hold at room temperature for 5 minutes.
9. Centrifuge at 4C for 10 minutes at 5500 rpm in Sorvall GSA rotor.
10. Remove as much supernatant as possible. Disperse pellet in 50 ml of 70% EtOH.
11. Centrifuge at room temperature for 5 minutes at 5500 rpm in Sorvall GSA rotor.
12. Remove as much supernatant as possible. Invert bottle on Kimwipe for 5 minutes to drain off 70%
EtOH. Dry pellet for about 5 minutes using gentle stream of filtered air. Do not dislodge pellet.

  

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

 

Collections: Biology and Medicine