Plasmid Maxiprep Protocol
1. Add 5 ml fresh O/N to 250-500 ml TB with salt and antibiotics.
2. Grow 16-24 hrs with shaking 37°C.
3. Pellet in large buckets 4000 RPM 10 min (Beckman JA 10) RT.
4. Remove all TB.
5. Resuspend the bacterial pellet from a 250 ml culture in 5 ml of solution I.
Solution I
50 mM glucose
25 mM Tris Cl (pH 8.0) Stored at 4°C
10 mM EDTA
6. Transfer to a 30 ml Oak Ridge tube. Let stand at room temperature for 5 minutes.
7. Add 10 ml of freshly made solution II. Cover the tube and mix the contents by shaking the tube several times. Let stand on ice for 10 minutes.
Solution II
0.2 N NaOH 0.6 ml 1ON NaOH
1% SDS 3 ml 10% SDS
26.4 ml H2O
8. Add 7.5 ml of an ice cold solution III. Mix the contents by inverting the tube several times. Let stand on ice for 10 minutes.
Solution III
5 M K acetate pH 4.8
To prepare solution III: To 60 ml of 5 M potassium acetate, add 11.5 ml of glacial acetic acid and 28.5 ml of H2O. The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate.
9. Spin 8500 RPM 20' 4°C Beckman J-17 fixed angle rotor.
10. Transfer the supernatant into 30 ml Oak Ridge tubes. Warm centrifuge to RT.
11. Add 0.6 vol. of isopropanol to each tube. Mix well and let stand at room temperature for 15 minutes.
12. Spin 8500 RPM J-13.1 swinging bucket 30' RT.
13. Wash x 1 with 70% EtOH. Spin same rotor 15'.
14. Lyophilize dry approx. 5'.
15. Resuspend in 4.7 ml TE 10' at 37°C.
16. Weigh 4.95 g exactly of cesium chloride into 13 ml Sarstedt tube.
17. Add: 4.5 ml of DNA in TE solution to cesium chloride and 450 ml of ethidium bromide.
18. Spin 8500 RPM 10' Beckman J-17 rotor at RT.
19. Fill quick seal tube - weigh and seal.
20. Spin Beckman ultracentrifuge VTI 65 rotor 55000 K O/N at 20°C or NVT 90 20°C 4 hrs.
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