Plasmid Miniprep

Materials

• 1–10 mL saturated bacterial culture
  • In general: 6 mL LB incubated overnight or 2 mL LB³⁰ incubated for 7–18 h should be sufficient for most purposes. For many sequencing reads on low-yield plasmids or some use else that requires a lot of DNA, it may be necessary to double the culture volume and lysis-neutralization steps, applying lysates to a single column.
    
  • 5–10 mL LB cultures for high- or low-copy plasmid, respectively, grown for 7–18 hr to saturation is generally sufficient to ≈saturate the spin column bed without introducing too much genomic DNA; or 2–3 mL LB³⁰ or TB cultures for the same. Too much cell mass can introduce genomic contamination.

  • A better culture medium for plasmid prep such as LB³⁰ or TB allows avoiding larger cultures needing another tube and centrifuge, while also inflating plasmid copy# due to the high yeast extract. 2 mL of such culture can be both pelleted and lysed in the same 2 mL microcentrifuge tube, simplifying the process.

• For each 1.5–2 mL culture: a 1.5 mL or 2 mL microcentrifuge tube (for both culture pelleting and miniprep)
  For each >2 mL culture:
  • a 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.
  • a 1.5 mL microcentrifuge tube (for miniprep reaction)
• Miniprep spin column (20–40 µg capacity) with collection tube.
  • Be sure not to use micro columns with small bed, 5 µg DNA capacity.
  • EconoSpin 1920 mini spin columns claim 30–40 µg DNA capacity, Quiagen QIAquick 20 µg, Zymo II/IIN 25 µg.
  • 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.

Buffers – must use aliquots; never directly use 500 mL stocks.

• Buffer P1 + RNase A, resuspension buffer.
  • Keep stored 2–8°C; make sure RNase added to the aliquot.
  • 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 ones with DNase activity (endA⁺).
• Buffer PE, wash buffer
  • For the original buffer bottle, make sure "Ethanol added" is checked on the lid.
  • Labmade PE is made by mixing: 20 mL filter-sterilized 0.5 M Tris-HCl pH 7.8, 180 g or mL sterile MilliQ water, and 800 mL 96–100% ethanol, made in detergent-free glassware.
• 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 clarified lysate to spin 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 2 min spin.
10. Elute DNA 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, 2 min in microcentrifuge for a looser pellet, more easily resuspendable. 3 min for blocks or tubes in a clinical centrifuge, usually with the max speed ≈4000×g.
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. Ensure no clumps remain.
3. Lysis: Add 250 μL Buffer P2 and mix immediately by inverting the tube 8–12 times until the solution becomes clear. Incubate 1–5 min. Do not allow lysis to proceed >5 min.
   • Qiagen protocol instructs 10–12 inversions and incubation for 5 min for maximal yield.
   • Zymo protocols instruct working in groups of ≤ten reactions so that there is not too long a lag between P2 addition and mixing.
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, which can be picked out with a tip.
   Centrifuge 15–30 s, ~20000×g, and discard the flow-through by aspiration.
   For vacuum manifold, apply vacuum until all columns are completely drained.
   • Aspirating the flow-through results in less buffer contamination of the DNA, as simply decanting it dirties the collection tube wall and lip with the buffers, which can then transfer to the spin column and elution tube it is eventually placed in.
7. (opt.) Wash the spin column by adding 250–500 µL Buffer PB.
   Centrifuge and drain, or vacuum, as in step 7.
   
   • Recommended for high-carbohydrate strains and high DNase activity (endA/dns⁺) strains.
   • Shyam washes with 250 µL PB for part plasmids as a precaution for maximizing life, and for high fluorescent protein-expressing cells to remove otherwise contaminating sfGFP.
8. Wash the spin column by adding 750 µL Buffer PE.
   Centrifuge and drain, or vacuum, as in step 7.
   •  Shyam uses 950 µL on vacuum manifold to reach the very top of the column. Too high for centrifuging, though.
9. Dry the column by centrifuging 2 min to remove residual wash buffer. (cannot be vacuumed)
   Immediately move to elution tubes, away from wash buffer ethanol vapor.
   
   • Allowing columns to rest in elution tubes for a few min, even heated in the same block as your eluent, may slightly improve elution by allowing residual ethanol to evaporate.
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, ≥15000×g. with open tube caps braced for rotation.
    
    • Warming the eluent, up to 60°C, can improve elution yield, especially of large plasmids.
    • Since the rotor turns counter-clockwise, face elution tube caps clockwise over adjacent holes if there is space, or against the bottom of the tube adjacent to the right, stacking the cap over the adjacent tube's cap if tightly packed. The caps must be braced for the rotation, not able to move, or else they will break off.
    • Centrifuging at a lower speed than step 6 may prevent tube caps from breaking. 
    • Store plasmid DNA at -20°C for tens of years, or even -80°C for longer-term archiving.

Cross-Contamination

Cross-contamination of samples and contamination of reagents with sample cells/DNA is essential quality control, without which one cannot be sure of any downstream usage of the purified plasmids in a set. It is easy to cross-contaminate samples if not being mindful to avoid it.

Care must be taken to not touch any surface that cells, DNA, or buffers come into contact with, as doing so among multiple samples can cross-contaminate them. This includes the underside of tube lids and the top edge of spin columns.

• When inoculating colonies into tubes with media, you may drop the tip with the colony in. If ejecting with a pipet, do not allow the pipet to touch the inside of the tube (or block). Clean the pipet shaft/ejector rod otherwise.
• When inoculating colonies into media in wells of a culture block, place the tip firmly into the well and rub the colony in a corner below the surface of the medium. When releasing a tip with your hand, take care that the tip does not stick to your hand or flick out, as a glove's static electricity can attract the tip you handle or neighboring tips. 
• Cell cultures must not seep out of the lids of overfilled tubes, nor splash while decanting the medium after pelleting.
• When using the same pipet tip for adding a buffer to multiple samples, hold the tip at least 1–1.5 cm (½ in) above the lip of the sample tube, and dispense in a stream pointed at the wall of the recipient tube, not straight down. This way, the stream force is reduced and addition to the sample doesn't produce splash or bubbles. Switch tips if you accidentally touch a sample tube or see splashback. Most stray liquid particles can be seen sputtering off the end of the pipet tip itself at the end of dispensing.
• The protruding ring on the inner side of tube lids must not touch one's gloves. It is easy to accidentally do so with closely spaced tubes while opening them sequentially, adding buffers to the mouths of the tubes, and closing them. Pay attention and be dexterous. Give clearance to the tube lids and space tubes apart.