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DNA Plasmid Maxiprep Protocol DNA Plasmid Maxiprep Protocol

1. Pick single colony and inoculate 250 ml of LB broth containing 100 mg/ml ampicillin or appropriate antibiotic. Shake at 250 RPM overnight.

2. Centrifuge cells in a Sovall GSA (250 ml)or SLA-3000 (500 ml) rotor at 5 k × g for 10 minutes.

3. Resuspend cell pellet in 10 ml of GTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8) and add a spatula tip of lysozyme powder. A good suspension is consistent without clumps of cell pellet.

4. Add 20 ml of NaOH/SDS lysis solution (0.2 M NaOH, 1% SDS). Use a spatula to stir and dissolve the cells until the solution becomes clear, yellow. This takes about 2 to 3 minutes and can be conducted at room temperature.

5. Add 15 ml 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. Sir and vortex to neutralize. Centrifuge at 10 k × g for 10 minutes.

6. Into a clean GSA bottle pour the supernatant through a small two-ply square of cheese cloth placed in the center of a funnel. The cheese cloth catches any fragments of SDS/genomic DNA pellet the is floating on the surface.

7. Precipitate the nucleic acids with 30 ml of isopropanol on ice for 10 minutes and centrifuge at 10 k × g for 10 minutes.

8. Aspirate off all the isopropanol supernatant. Dissolve the pellet in 5 ml of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5). Transfer to a clean SS-34 tube. Some nucleic acid pellet will be stuck on the sides of the bottle; thoroughly dissolve all the pellet.

9. Add 5 ml of 5 M LiCl solution to precipitate RNA. Leave on ice for 10 minutes and centrifuge at 10 k × g for 10 minutes.

10. Pour off the supernatant containing plasmid DNA into a clean SS-34 tube. Add an equal volume of isopropanol (10 ml) and precipitate the nucleic acids on ice for 10 minutes. Centrifuge at 10 k × g for 10 minutes.

11. Aspirate off all the isopropanol supernatant. Dissolve the pellet in 1 ml of TE buffer. Transfer TE solution into a 1.5 ml epindorf tube. Add 15 ml of RNAse A solution (20 mg/ml stock stored at -20 °C), vortex and incubate at 37 °C for 20 to 30 minutes to digest remaining RNA.

12. Precipitate the plasmid DNA with PEG solution (30% polyethylene glycol, 1.6 M NaCl) by adding 0.4 ml and incubating overnight on ice for full recovery. This step discriminates very large plasmid DNA from small nucleic acid fragments as only the larger plasmid DNA precipitate.

13. Aspirate off the supernatant PEG buffer and dissolve the PEG pellet in 0.4 ml of TE buffer.

14. 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.

15. 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.

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

17. Aspirate off ethanol solution and resuspend or dissolve DNA pellet in 0.3 to 0.5 ml of TE buffer. This is the final stock of PEG pure plasmid DNA which is suitable for DNA sequencing and long term storage.

18. Measure the concentration of the plasmid DNA by diluting stock into water at 1:200 or 5 ml of DNA per 1 ml of water (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 mg/ml/OD 260nm).