Mini-Prep Protocol for Recovery of Plasmid DNA

1. Grow cultures overnight in 5 ml L-broth supplemented with the appropriate antibiotics at 37°C with vigorous shaking (200-250 rpm).

2. Transfer cultures to centrifuge tubes and centrifuge at 3,000 rpm for 10 minutes (RT or 4°C). Discard supernatant.

3. Resuspend pellets in 180 µl Solution I (Tris-glucose-EDTA, pH 8.0) by vortexing.

4. Transfer suspension to microcentrifuge tubes containing 20 µl lysozyme (prepared fresh, 10 mg/ml in Solution I).

5. Vortex quickly, incubate tubes at room temperature for 5 minutes.

6. Add 0.4 ml Solution II (prepared fresh - combine 2 ml 1 M NaOH and 1 ml 10% SDS, bring to final volume of 10 ml with sdH2O). Cap tubes and mix contents by inverting several times (do not vortex).

7. Incubate tubes on ice for 5 minutes.

8. Add 0.3 ml refrigerator-cold Solution III (potassium acetate-acetic acid). Mix by inverting and gentle vortexing.

9. Incubate tubes on ice for 10 minutes.

10. Microfuge tubes at room temperature on high for 5 minutes.

11. Transfer supernatant to new tubes and repeat microfugation for 5 minutes.

12. Transfer supernatant to new tubes. Add 0.5 ml (0.6 volumes) isopropanol. Mix well by inverting and vortexing.

13. Microfuge tubes on high at room temperature for 5 minutes. Discard supernatant.

14. Wash pellet by slowly adding 0.5 ml 70% ethanol. It is not necessary to disturb pellet for this wash. Carefully pour off 70% ethanol and discard.

15. Dry pellets at room temperature for 10 to 15 minutes inverted in rack over paper towels.

16. Gently resuspend pellet in 100 µl 1x TE buffer.

17. Add 2 µl RNAse (10 mg/ml). Incubate tubes at room temperature for 15 minutes.

18. Add 100 µl buffered phenol (use bottom layer). Mix by inverting and by vortexing 10 seconds.

19. Microfuge on high at room temperature for 3 minutes. Transfer top aqueous phase to new tubes, avoiding interface. Discard organic phase.

20. Add 100 µl chloroform:isoamyl alcohol (24:1), vortex for 5 seconds to mix. Microfuge tubes for 1 minute.

21. Transfer top aqueous phase to new tubes. Add 15 µl 3 M sodium acetate (pH 4.5-5.5). Mix thoroughly by inverting and by vortexing. Place tubes on ice.

22. Add 250 µl (2.5 volumes) of freezer-cold (-20°C) 95% ethanol. Vortex to mix. Allow DNA to precipitate overnight at -20°C or for 30 minutes at -70°C.

23. Microfuge tubes for 15 minutes to pellet DNA. Gently pour off supernatant and discard.

24. Dry pellets completely in rack inverted over paper towels. Resuspend pellets in 30 µl 1x TE buffer. Store at -20°C.

1. EDTA contained in solution I aids lysozyme in degradation of cell wall (step 3).

2. Alkaline-SDS lysis (step 6) must be done gently to avoid contamination with chromosomal DNA. The SDS lyses the cells and the NaOH denatures proteins and chromosomal (linear) DNA. Optimal pH of mixture at this point is between 12.0 and 12.6.

3. Addition of solution III (step 8) readjusts pH to neutral (solution III contains acetic acid) and its high salt content (3 M in potassium acetate) precipitates SDS-protein complexes, RNA, and chromosomal DNA.

4. Addition of isopropanol (step 12) precipitates nucleic acids, 70% ethanol wash (step 14) removes traces of isopropanol remaining.

5. Phenol extraction (step 18) removes remaining proteins and linear DNA.

6. Chloroform extraction (step 20) removes carbohydrates, remaining proteins, and residual phenol. The one-part isoamyl alcohol reduces foaming.

  Solution I (500 ml)              Solution II (100 ml)          Solution III (100 ml)                                                                                        4.50 g glucose (50 mm)           0.8 g NaOH (0.2 N)            29.5 g potassium acetate      1.97 g Tris-Cl (25 mm)           1.0 g SDS (1%)                11.5 ml glacial acetic acid  1.86 g EDTA (10 mm)                                                                                                                                                         Disolve reagents in 300          Dissolve reagents in a        Dissolve reagents in a        ml dH2O.  Adjust pH to           final volume of 100 ml        final volume of 100 ml.       8.0 with HCl.  Adjust            dH2O.  Prepare fresh,         Sterilize by autoclaving.     volume to 500 ml.                just before use.                                          Sterilize by autoclaving.                                                                                                                                                                                                                                         3 M sodium acetate (100 ml)      10x TE Buffer (500 ml)        LB Broth (1 L)                                                                                        40.8 g sodium acetate            7.88 g Tris-Cl                10.0 g Bacto-tryptone                                          1.86 g EDTA                    5.0 g Bacto-yeast extract                                                                                      10.0 g NaCl                                                                                                           Dissolve in approximately        Dissolve reagents in 350      Dissolve reagents in 950      50 ml dH2O.  Bring to            ml dH2O.  Adjust to pH        ml dH2O.  Adjust pH to        final volume of 100 ml.          8.0 with HCl.  Bring to       7.0 with 5 N NaOH (~0.2      Sterilize by autoclaving.        final volume of 500 ml.       ml).  Adjust volume to                                         Sterilize by autoclaving.     1000 ml.  Sterilize by                                         Dilute 1:10 before use.       autoclaving.