Boil-E miniprep protocol

1. Collect Bugs:
   

1. 1.Pour 2ml overnight culture into 1.5ml centrifuge tube
   

2. 2.Spin 30 secs; pour off supernatant
   

3. 3.Resuspend in remaining broth by vortexing
   

1. Extracting Plasmid:
   

4. 4.Make fresh STET/lysozyme solution
   •  STET: 8% sucrose + 50mM Tris8 + 50mM EDTA + 5% TritonX-100
   •  can store at room temperature
   •  Add fresh lysozyme powder to 1mg/ml final concentration
   

5. 5.Vortex in 400µl STET/lysozyme solution
   •  remember: have large vortex holder
   

6. 6.Boil 1’
   

7. 7.Spin 10’ @ R.T.
   

8. 8.Remove pellet with toothpick or yellow tip
   

1. Cleaning up Plasmid:
   

9. 9.Add 400µl chilled isopropanol; vortex.
   

10. 10.Spin 15’ @ 4°C.
    

11. 11.Wash pellet with 70% EtoH.
    

12. 12.Lyophilize.
    

13. 13.Resuspend in 50-100µl TE8. Use 1µl per miniprep.
    

EasyPrep

1. 1.Centrifuge 1.5 ml of bugs for 20-30 secs.
   

2. 2.Resuspend in 50 µl of lysis buffer
   •  10mM Tris-8 + 1mM EDTA + 15% sucrose + 2mg/ml lysozyme + 0.2 mg/ml RNase + 0.1 mg/ml BSA
   •  store at -20°C
   

3. 3.Boil 1 min.
   

4. 4.Chill 1 min.
   

5. 5.Spin 15-20 min @ R.T.
   •  Can leave pellet @ bottom and draw from supernatant. Remove pellet for longer-term storage.
   

6. 6.Use 3µl-5µl for digestion.
   

© 2008 Mount Sinai Medical Center