DNA?Plasmid?Miniprep?Protocol

1. Pick single colony and inoculate 5 ml of LB broth containing 200 g/l ampicillin or 1mg/5ml. Optional: Use a 15ml conical tube with a loosened cap and a piece of tape to hold it in place. Shake at 250 RPM 37℃ overnight.

2. Centrifuge 1.5mL cells in 1.5 mL Eppendorf tube at top speed for 1 minute. Aspirate supernatant.

3. Resuspend cell pellet in 100 μl of GTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8).  Vortex gently if necessary.

4. Add 200 μl of NaOH/SDS lysis solution (0.2 M NaOH, 1% SDS). Invert tube 6-8 times.

5. IMMEDIATELY add 150 μl of 5 M potassium acetate solution (pH 4.8) . This solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in an insoluble white, rubbery precipitate. Spin at top speed 1 min.

6. Transfer supernatant to new tube, being careful not to pick up any white flakes.  Precipitate the nucleic acids with 0.5mL of isopropanol on ice for 10 minutes and centrifuge at top speed for 1 minute.

7. Aspirate off all the isopropanol supernatant. Dissolve the pellet in 0.4 ml of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5). Add 10ul of RNAse A solution (20 mg/ml stock stored at -20 ℃), vortex and incubate at 37 ℃ for 20 to 30 minutes to digest remaining RNA.

8. Extract proteins from the plasmid DNA using PCIA (phenol/chloroform/isoamyl alcohol) by adding about 0.3 ml. Vortex vigorously for 30 seconds. Centrifuge at full speed for 5 minutes at room temperature. Note organic PCIA layer will be at the bottom of the tube.

9. Remove upper aqueous layer containing the plasmid DNA carefully avoiding the white precipitated protein layer above the PCIA layer, transferring to a clean 1.5 ml epindorf tube.

10. Add 100 m l of 7.5 M ammonium acetate solution and 1 ml of absolute ethanol to precipitate the plasmid DNA on ice for 10 minutes. Centrifuge at full speed for 5 minutes at room temperature.

11. Aspirate off ethanol solution and resuspend or dissolve DNA pellet in 50μl of DNA . Dissolve 5μl in 995μl of water, and spec (blank spectrophotometer to water). The absorbance at 260 nm multiplied by ten is the concentration of the DNA in units of mg/ml for a 1 cm pathlength cuvette (i.e. 50 m g/ml/OD 260nm).