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.

• Qiagen 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.
• Qiagen Buffer P2, lysis buffer
  • Check for crystallization; resuspend by vortexing.
• Qiagen Buffer N3, neutralization buffer
  • Check for crystallization; resuspend by vortexing.
• Qiagen Buffer PB, optional wash buffer
  • Recommended for high-carbohydrate strains and ones with DNase activity (endA⁺).
• Qiagen 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 EB / DNA-grade Tris buffer / TE, or nuclease-free deionized water.
  • Elution buffer ensures optimal pH for DNA elution from silica membrane, solvates DNA better than water, 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 low 0.1 mM EDTA over the common 1 mM, as 1 mM may be enough to inhibit downstream Mg²⁺-dependent enzymatic reactions. Buffer must be DNA-grade.
  • For electrotransformation, DNA in elution buffer does not seem to contribute enough salt to cause arcing or reduce τ. Still, using large DNA volumes in library transformation efficiency might still benefit from elution in water.
    
  • Heat the eluent only before use to preserve elution buffer and plastic integrity.

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 100–500 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.
   • ≥10000×g, 30 s for ≤2 mL cultures in microcentrifuge.
   • 5000×g, 2 min in microcentrifuge for a looser pellet, easily resuspendable by vortexing in the next step.
   • For blocks or larger tubes in a centrifuge, 3 min around the max speed ≈4000×g. Blocks can be quickly inverted and shaken to remove medium, and the block surface blotted, without noticeable cross-contamination of DNA. Alternatively, aspirate medium from each well without needing to change aspirator tips.
2. Resuspend cell pellet in 250 μL Buffer P1 by vortexing or pipetting, ensuring no clumps remain. Transfer to a microcentrifuge tube if pelleted in a larger tube or block. 
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 10–12 min, ≥18000×g.
   • Try not to shorten this time, as it is when RNase A 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 alcohol 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 if it flows into the column.
   Centrifuge 15–30 s, ≥18000×g, and discard the flow-through by aspiration.
   or Apply vacuum to manifold until all columns are completely drained.
   • Instead of decanting, aspirating collection tube contents prevents flow-through contaminants from spreading onto the side of the column and contaminating the final elution tube.
7. (opt.) Wash the spin column by adding 100–500 µL Buffer PB.
   Centrifuge and drain, or vacuum, as in step 6.
   
   • Recommended for high-carbohydrate strains and DNase activity (endA⁺) strains.
   • Shyam washes with 100 µL PB for part plasmids as a precaution to maximizing the sample life, and for superfolder fluorescent protein-expressing cultures, whose fluorescent protein otherwise contaminates the DNA (seemingly inconsequentially for its use).
8. Wash the spin column by adding ≥750 µL Buffer PE.
   Centrifuge and drain, or vacuum, as in step 6.
   •  Shyam uses 950 µL on vacuum manifold to reach the very top of the column and wash the groove, though too high for centrifuging.
   • Two–three sequential smaller washes, e.g. 400 µL, may be more effective than one at minimizing contaminants for sensitive applications. These can be pipetted pointing at opposite sides of the column.
9. Dry the column by centrifuging 2 min to remove residual wash buffer. (cannot be vacuumed) Meanwhile, you may preheat eluent.
   Then immediately, quickly separate and discard collection tube and place the column in the remaining fresh, labeled 1.5 mL tube.
   
   • Residual ethanol in matrix inhibits elution. 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 25–75 μL elution buffer EB or water, opt. heated, 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.
    Check column and tube labels match, and discard column.
    
    • Prefer an elution buffer to water; read notes in Materials. Plasmids should, by lab standard, be eluted and stored in a protective buffer.
      
    • Warming the eluent, up to 60°C, can improve elution yield, especially of large plasmids.
    • Since the rotor turns counterclockwise, 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 4° temporarily, at -20°C for tens of years, or -80°C for longer-term archiving.
      

• DNA concentration and purity can be estimated using a NanoDrop™ spectrophotometer's microvolume pedestal using only 1–1.5 µL sample or TECAN NanoQuant Plate. It can show presence of high levels of typical salt contaminants (especially guanidinium) via the 260/230 nm absorbance ratio. See Microvolume Spectrophotometry.
• Store purified DNA at 4° for short-term use and −20°C for longer-term. Thaw briefly at room temp and (for best practice) keep on ice or 1°C block during usage. Linear DNA is more prone to degradation at ends than circular plasmid DNA. Cohesive (recessed, "sticky") ends are rather unstable and should be used in assemblies soon after generating and purifying, as the ends are not very stable for long periods even when frozen.

Cross-Contamination

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

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. In a miniprep, this includes the inside of tube lids and the outer surface 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.
• 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 in the rack.
  Be especially vigilant when the lids are wet with sample, as when handling open tubes after lysis and neutralization.
• Avoid touching the lower part of spin columns, so as to avoid cross-contamination of the elution tube it will later contact. This is why it is a suggestion to not decant collection tubes, as flow-through wets the sides, spreads to the spin column when it is reinserted, and can spread to gloves when handling or to the final elution tube.
• 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 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, which isn't contaminating.