Plasmid Miniprep

Materials

• 1–6 mL saturated bacterial culture
  • A maximum of 6 mL LB culture or 2 mL LB³⁰ / TB cultures grown for 7–18 hr to saturation is generally sufficient to saturate the spin column with medium- to high-copy plasmid DNA without introducing much genomic DNA and salt contamination.
  • A 3–4-fold smaller but decent yield of low-copy plasmids can be obtained in the same volume LB³⁰ or TB
• For each 1.5–2 mL culture: 1.5 mL or 2 mL microcentrifuge tube (for both culture pelleting and miniprep)
• For each >2 mL culture:
  15 mL conical centrifuge tube (for culture pelleting), and…
  • Cultures can also be pelleted iteratively or in parallel in 1.5 or 2 mL microcentrifuge tubes.
  1.5 mL microcentrifuge tube (for miniprep reaction)
• Miniprep spin column with collection tube
  • Using a vacuum manifold, collection tubes are only used for drying columns (step 10), and thus may be reused, with fresh ones saved if needed.
  • Econospin columns have a 30–40 µg capacity (50 µL of 600–800 ng/µL)
• Buffer P1 + RNase A, resuspension buffer.
  • Keep stored 2–8°C; make sure "RNase added" is checked on the lid.
  • 100 mg/L RNase A vials are 1000×; add 50 µL to a typical 50 mL aliquot of Buffer P1.
• Buffer P2, lysis buffer
  • Check for crystallization; resuspend by vortexing.
• Buffer N3, neutralization buffer
  • Check for crystallization; resuspend by vortexing.
• Buffer PB, optional wash buffer
  • Recommended for high-carbohydrate strains and high DNase activity (endA⁺) strains.
• Buffer PE, wash buffer
  • For the original buffer bottle, make sure "Ethanol added" is checked on the lid.
  • Labmade PE is made by mixing: (INSERT INSTRUCTIONS FROM BOTTLE)
• Eluent: elution buffer (Qiagen Buffer EB or TE), or nuclease-free deionized water.
  • Elution buffer ensures optimal pH for DNA elution from silica membrane and maintains an alkaline pH that protects DNA from hydrolysis and degradation from local pH extremes during freeze-thaw cycles.
  • Qiagen elution buffer is 10 mM Tris-Cl pH 8.5. TE buffer (Tris-Cl, EDTA) pH 8–9 is also a common elution buffer, adding EDTA, whose purpose is to protect DNA from contaminating nucleases by chelating the necessary Mg²⁺ cofactor. Prefer TE with 0.1 mM EDTA over 1 mM, as the latter may be enough to inhibit downstream Mg²⁺-dependent enzymatic reactions.
  • ≤0.5 µL DNA in elution buffer does contribute enough salt to cause arcing during electroporation.
    DNA should be eluted in water if intended for electroporation with a larger DNA volume.

Summary

1. Pellet culture.
2. Resuspend in 250 µL P1.
3. Lyse with 250 µL P2; invert.
4. Neutralize with 350 µL N3; invert.
5. Clarify lysate with a spin.
6. Bind lysate to column; spin/vacuum.
7. (opt.) Wash column with 200–500 µL PB; spin/vacuum.
8. Wash column with 750 µL PE; spin/vacuum.
9. Dry column with a spin.
10. Elute in ~50 µL EB or water; spin.

Procedure

1. Pellet saturated cultures by centrifugation. Decant away supernatant medium.
   • 20000×g, 30 s for ≤2 mL cultures in microcentrifuge.
   • ≈5000×g, 3 min in clinical centrifuge or 2 min in microcentrifuge, for a looser pellet, more easily resuspendable.
2. Resuspend pelleted cells in 250 μL Buffer P1 by pipetting or gentle vortexing; transfer to a microcentrifuge tube if pelleted in a larger tube.
3. Lysis: Add 250 μL Buffer P2 and mix immediately by inverting the tube 6 times until the solution becomes clear. Do not allow lysis to proceed >5 min (or 2.5 min to be safer).
4. Neutralization: Add 350 μL Buffer N3 and mix immediately by inverting the tube 6 times.
5. Clarify lysate: Centrifuge 10 min, 20000×g.
   • Try not to shorten this time, as it is when RNase digests RNA.
   Meanwhile, label spin columns. Set up columns on vacuum manifold, if using. Label elution tubes.
   • Spin columns are best labelled on the side of the column at the very top, as the lower side and the top protruding lip can be exposed to the alcohols in the wash buffers and erase the labels.
   • Hold the body of the vacuum manifold valves while turning them, as they are prone to breaking.
6. DNA–matrix binding: Decant or pipette the supernatant from step 5 into the labelled spin column set inside a collection tube or upon a vacuum manifold.
   • Take care not to dislodge pellet matter.
   Centrifuge 15–30 s, ~20000×g, and discard the flow-through by decanting or aspiration.
   For vacuum manifold, apply vacuum until all columns are completely drained.
   • Aspiration can result in less buffer contamination of the DNA, as decanting dirties the collection tube wall and lip, which can then transfer to the spin column and elution tube.
7. (opt.) Wash the spin column by adding 500 µL Buffer PB.
   Centrifuge and drain, or vacuum, as in step 7.
   
   • Recommended for high-carbohydrate strains and high DNase activity (endA⁺) strains.
   • Shyam washes with 200 µL PB for part plasmids, just as a precaution for maximizing shelf-life.
8. Wash the spin column by adding 750 µL Buffer PE.
   Centrifuge and drain, or vacuum, as in step 7.
9. Dry the column by centrifuging 2 min to remove residual wash buffer. (cannot be vacuumed)
10. Elute the DNA by adding 30–75 μL elution buffer EB or water to the center of the column matrix. Let stand for 1–4 min.
    Centrifuge 30 s, ≥10000×g.
    
    • Warming the eluent, up to 60°C, can improve elution yield, especially of large plasmids.
    • Face elution tube caps rightward over adjacent holes if there is space, or against the bottom of the adjacent right tube, stacking the cap over the adjacent cap if tightly packed.
    • Centrifuging at a lower speed than step 7 may prevent tube caps from breaking. 
    • Store plasmid DNA at -20°C for many years, or even -80°C for longer-term archiving.