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Plasmid midi-prep from bacteria











Last Update: December 2006
Based on a protocol developed by Michael Feldbrugge
1. Glycerol mix (1 L): Weigh 25 grams (liquid weight) of glycerol and add dH2O to 1 Liter (Autoclave)
2. Potassium mix (1 L): Mix 170 mL of 1M KH2PO4 (monobasic), 720 mL of 1M K2HPO4 (dibasic), and 110 mL of dH2O (Autoclave)
3. TE50/10 (100 mL): Mix 5 mL of 1M Tris pH 7.5, 2 mL of 0.5M EDTA pH 8.0, and 93 mL of dH2O
4. TE10/1 (100 mL): Mix 1.0 mL of 1M Tris pH 7.5, 200 mL of 0.5M EDTA pH 8.0, and 99 mL of dH2O
5. 4M LiCl (100 mL): Mix 17 g of LiCl with 100 mL of dH2O (Autoclave)
6. TB media (1 L): Mix 12 g of Bacto Tryptone, 24 g of Bacto Yeast Extratct, and dH2O to 900 mL. Autoclave (add 100 mL of Glycerol mix (after autoclave)
7. Lysis solution (30 mL): Mix 1.5 mL of 20% SDS, 1.2 mL of 5M NaOH, and 27.3 mL of dH2O
8. 3M KOAc pH 4.8 (100 mL) Mix 29.4 g of potassium acetate, 40 mL of dH2O, add Glacial acetic acid to pH 4.8, and dH2O to 100 mL (Filter sterilize) (store at 4oC)
9. 1M KH2PO4 (100 mL): Mix 13.6 g of KH2PO4 (monobasic), and 100 mL dH2O (Autoclave)
10. 1 M K2HPO4 (100 mL): Mix 17.42 g of K2HPO4 (dibasic), and 100 mL dH2O (Autoclave)
1. In a 125 mL flask, add: 36 mL of TB media + 4 mL of Potassium mix + applicable antibiotic
2. Transfer a single colony to the media and grow overnight
3. Spin ~20 mL of culture in a Sarstedt tube at 6,000 rpms for 10 mins (4oC)
4. Decant supernatant and resuspend pellet in 1.5 mL of TE50/10 + 4 mL of 21 mg mL-1 RNAse
5. Incubate on ice for 5 mins and add 3 mL of Lysis solution
6. Incubate on ice for 10 mins and add 2.25 mL of 3 M KOAc pH 4.8
7. Incubate on ice for 10 mins and centrifuge at 10,000 rpms for 15 mins (4oC)
8. Transfer supernatant to new tube containing 3 mL of 2-propanol and invert to mix (optional: leave ON)
9. Centrifuge at 10,000 rpms for 30 minutes and discard supernatant
10. Let pellet drain and resuspend in 300 mL TE10/1. Trasfer solution to a 1.5 mL tube
11. Add 300 mL 4M LiCl and incubate on ice for 15 mins
12. Centrifuge for 15 mins at full speed (4oC) and transfer supernatant to a 2 mL tube
13. Fill the tube with 100% EtOH (~1.5 mL), mix by inversion (or: leave at 4oC ON) and centrifuge for 15 mins at full speed (4oC)
14. Resuspend in 400 mL of TE10/1 + 5 mL of 21 mg mL-1 RNAse and incubate for ~30 mins at 37oC
15. Extract with phenol: add 400 mL of TE-phenol, mix by vortexing, warm at 37oC for ~1 min, mix again, and centrifuge at full speed for 3 mins
16. Transfer upper transparent phase to a 1.5 mL tube, add 400 mL of 24 Chloroform: 1 isoamyl alcohol and carefully mix by vortexing
17. Centrifuge at full speed for 20 secs and transfer clear upper phase to a 1.5 mL tube.
18. Add 40 mL of 3M NaOAc (1/10 vol) and ~1 mL of 100% EtOH (to the top of the tube) and mix by inversion (optional: leave ON)
19. Centrifuge at top speed for 15 mins and aspirate off all the supernatant
20. Dry pellet in the SpeedVac for 5 mins.
21. Resuspend in 400-500 mL TE10/1. The DNA sample is ready for downstream applications.
22. Determine concentration spectrophotometrically (~20 mL of culture should produce ~0.5-1.0 mg of plasmid DNA from a high copy plasmid)
23. Optional: determine the amount that is barely visible on a THIN EtBr-agarose gel
Note: EtBr has a visibility limit of about 100-200 ng on agarose gels. Thick gels will mask some of the signal, specially at low DNA concentrations