Plasmid midi-prep from bacteria

PLASMID MIDI-PREP FROM BACTERIA

Last Update: December 2006

Based on a protocol developed by Michael Feldbrugge

PREPARE SOLUTIONS

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. TE_50/10 (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. TE_10/1 (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 4^oC)

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)

PROCEDURE

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 (4^oC)

4. Decant supernatant and resuspend pellet in 1.5 mL of TE_50/10 + 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 (4^oC)

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 TE_10/1. 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 (4^oC) and transfer supernatant to a 2 mL tube

13. Fill the tube with 100% EtOH (~1.5 mL), mix by inversion (or: leave at 4^oC ON) and centrifuge for 15 mins at full speed (4^oC)

14. Resuspend in 400 mL of TE_10/1 + 5 mL of 21 mg mL^-1 RNAse and incubate for ~30 mins at 37^oC

15. Extract with phenol: add 400 mL of TE-phenol, mix by vortexing, warm at 37^oC 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 TE_10/1. 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