PLASMID MIDI-PREP FROM BACTERIA
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1154
| PREPARE SOLUTIONS | |
| 1. Glycerol mix (1 L): | Weigh 25 grams (liquid weight) of glycerol and add dH2 O to 1 Liter (Autoclave ) |
| 2. Potassium mix (1 L): | Mix 170 mL of 1M KH2 PO4 (monobasic), 720 mL of 1M K2 HPO4 (dibasic), and 110 mL of dH2 O (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 dH2 O |
| 4. TE10/1 (100 mL): | Mix 1.0 mL of 1M Tris pH 7.5, 200 m L of 0.5M EDTA pH 8.0, and 99 mL of dH2 O |
| 5. 4M LiCl (100 mL): | Mix 17 g of LiCl with 100 mL of dH2 O (Autoclave ) |
| 6. TB media (1 L): | Mix 12 g of Bacto Tryptone, 24 g of Bacto Yeast Extratct, and dH2 O 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 dH2 O |
| 8. 3M KOAc pH 4.8 (100 mL) | Mix 29.4 g of potassium acetate, 40 mL of dH2 O, add Glacial acetic acid to pH 4.8, and dH2 O to 100 mL (Filter sterilize) (store at 4o C) |
| 9. 1M KH2 PO4 (100 mL): | Mix 13.6 g of KH2 PO4 (monobasic), and 100 mL dH2 O (Autoclave ) |
| 10. 1 M K2 HPO4 (100 mL): | Mix 17.42 g of K2 HPO4 (dibasic), and 100 mL dH2 O (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 (4o C) | |
| 4. Decant supernatant and resuspend pellet in 1.5 mL of TE50/10 + 4 m L 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 (4o C) | |
| 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 m L TE10/1 . Trasfer solution to a 1.5 mL tube | |
| 11. Add 300 m L 4M LiCl and incubate on ice for 15 mins | |
| 12. Centrifuge for 15 mins at full speed (4o C) and transfer supernatant to a 2 mL tube | |
| 13. Fill the tube with 100% EtOH (~1.5 mL), mix by inversion (or: leave at 4o C ON) and centrifuge for 15 mins at full speed (4o C) | |
| 14. Resuspend in 400 m L of TE10/1 + 5 m L of 21 mg mL-1 RNAse and incubate for ~30 mins at 37o C | |
| 15. Extract with phenol: add 400 m L of TE-phenol, mix by vortexing, warm at 37o C 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 m L 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 m L 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 m L 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 |
