Gel Purification

Materials

• Electrophoresed gel
• Transparent tray/dish for cutting surface (e.g. petri dish)
• Blade, scalpel, or coverslip
• Two 1.5 mL microcentrifuge tube per sample to purify
  • 5–10 bands can be molten in one tube (3 µL buffer / mg gel fragment), if so much gel is needed to obtain desired amount of particular DNA.
  • Label the tubes with sample names. One will be used for gel melting and one for the product.
• DNA purification column. Often low, 5 µg-capacity from smaller silica bed to allow lower elution volume and thus higher DNA concentration.
  • Zymo-Spin I column: 5 µg DNA/RNA, ≥6 µL eluent, 800 µL capacity
  • Epoch Econospin micro column 3010-250: ≥5 µL eluent, 800 µL capacity. Includes collection tube.
  • Epoch Econospin mini column 1910-250: 30–40 µg DNA, ≥10 µL eluent, 800 µL capacity. Includes collection tube.
• Collection tube, used to collect column flow-through waste.
• Qiagen Buffer QX1, an agarose dissolving buffer (≥ 3 µL/mg gel fragment)
  Or Qiagen Buffer QG or Zymo Agarose-Dissolving Buffer, with modified disposal and optimization protocols. See next section for comparison.
• 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.
  • 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.
• Gel viewing equipment
  • Blue light transilluminator, orange shield/glasses for blue/green dyes (GelGreen)
  • UV transilluminator and UV shield for UV dye (EtBr, GelRed)
• 42–55°C heating block compatible with 1.5 mL tubes
• Microcentrifuge
• (opt.) Vacuum manifold and vacuum source, to speed column loading steps is vacuum pressure is sufficiently strong.

Choice of agarose-dissolving buffer

• Qiagen has a newer agarose dissolving buffer called QX1 that ensures no guanidinium salt contamination because it uses a different chaotropic salt (sodium phosphate) that leaves your column contaminant-free. Presumably the new salts wash away more readily than guanidinium). Josh Atkinson learned to use QX1 from the Ajo-Franklin Lab when at LBNL and brought word to me.
• Qiagen Buffer QG and presumably Zymo ADB: 5.5 M guanidinium thiocyanate (GuSCN), 20 mM Tris HCl, pH 6.6
  Qiagen Buffer QX1: 7 M NaClO₄ , 10 mM NaAc, pH 5.3
• QX1 comes from the Qiagen QIAEX II kit, which uses silica beads instead of columns, but by simply using separately-purchased QX1 (discontinued) in place of Qiagen QG or Zymo ADB, QX1 users recover just as much DNA while never seeing a guanidinium contaminant peak upon Nanodrop analysis of the gel-purified DNA.
• Furthermore, because bleach-treating thiocyanate releases cyanide gas, eliminating QG/ADB allows all column purification wastes (with miniprep, PCR purification) to be single-stream and bleached for simple drain-disposal.
• Buffer QG/ADB must be collected separately if used, so as to not be bleached with the majority of liquid waste. The QG/ADB waste must be ultimately collected by EHS.
• In the QX1-adapted gel purification protocol, washing the column twice with 200 µL PE was found to be sufficient for DNA that assembles well in our experience, though you may use 2×500 µL. Two washes are theoretically more effective at removing the salts, thus prescribed with QG/ADB protocols. A single 500–750 µL wash may also be sufficient. Residual QX1 salt contamination in DNA is hard to measure, as its salts don't have strong absorbance like guanidinium does.
• Though the QIAEX II procedure prescribes a primary 500 µL wash with QX1 to wash away residual agarose and two 500 µL washes with PE, this may only be necessary for its intended QIAEX II kit, which requires pipetting washes off the top of a silica pellet, a procedure that would seem to be more prone to contaminant carryover and thus require larger and sequential washes.

Summary

1. Cut gel bands into tubes and opt. weigh gel fragments.
2. Dissolve in ~400 µL or 3 volumes QX1/QG,
   Incubated 42–55°, ~10 min, vortexed.
3. Bind DNA to micro spin column; spin/vacuum.
4. Wash column with 500 µL PE; spin/vacuum.
5. Dry column with a 2 min spin.
6. Elute DNA in 5–15 µL EB or water; spin.

Procedure

1. Visualize DNA on gel placed on tray/dish over blue transilluminator. Capture image for later reference, as excision will destroy the gel.
2. Excise desired bands precisely with blade, and scrape into 1.5 mL tube. Position cuts on the four sides of each band. Minimize contact with other bands and including excess agarose devoid of visible DNA. If desired band is close to undesired ones, be more conservative in cut placement to reduce capture of undesired DNAs.
   • The less agarose in solution, the more efficient the purification will be. Larger fragments take longer to melt and require more dissolving buffer, which introduces more salts, etc to the purification. Dissolving fragments >200 mg will not fit with 3× dissolving buffer in the columns all at once.
3. (opt.) Weigh tubes for gel fragment masses, with an empty tube tared.
4. Dissolve: Add 3 volumes Buffer QX1, QG, or ADB per volume gel (3 µL per milligram gel) to each sample, and ensure gel fragments are in buffer, or else centrifuge or knock them down into it. For >2% agarose gels, use 6 µL.
   _{For QX1, salt concentration impacts DNA fragment affinity to silica according to size, which can optionally be adjusted to maximize yield:}
   • _{Fragments <100 bp: 6 volumes QX1}
   • _{Fragments 100–4000 bp: 3 volumes QX1 (found to work well enough for other conditions, too)}
   • _{Fragments >4000 bp: 3 volumes QX1 + 2 volumes water}
   • _{All fragments in >2% agarose gels: 6 volumes QX1}
   Incubate at 42–50°C 5–10 min to melt the gel fragments, vortexing vigorously a few times intermittently.
   Hold up to the light; look for any refraction at the buffer surface to indicate unmolten gel remaining.
   • For QX1 or QG, the color should be yellow, indicating low enough pH for silica binding. If orange or purple, add 10 µL 3 M NaOAc pH 5 (kept in a tube in the cabinet).
5. (opt.) iPrOH: If using Buffer QG or ADB, add 1 volume isopropanol (1  µL/mg gel) for higher yields of DNA fragments <500 bp and >4 kb. Vortex to mix.
6. (opt.) Cool: Allow samples to cool a minute to room temp to enhance DNA binding to silica
7. DNA–matrix binding: Pipette or pour dissolved gel samples into low-capacity "micro" columns to bind DNAs to matrix. 
   Centrifuge ≥18,000×g, 30 s. Or apply Vacuum to columns set in vacuum manifold until all liquid has passed through.
   • Strong vacuum pressure is needed for the viscous solution, often requiring centrifuging instead.
   • Pipetting can ensure undissolved fragments will be noticed by clogging the tip. And it can allow you to apply the sample to the bottom of the column and minimize contamination of the column walls.
8. (rare, opt.) Pre-Wash: Add 500 µL Buffer QG or ADB to column, and similarly centrifuge or apply vacuum. Empty collection tube.
   • Omit for most purposes. "Only required when the DNA will subsequently be used for salt-sensitive applications, such as blunt-end ligation, direct sequencing, in vitro transcription, or microinjection."
9. 2×Wash: Add 200–300 µL wash buffer PE to column, and similarly centrifuge or apply vacuum. Repeat once. Empty the collection tube. Or use a single 500–750 µL wash.
   
   • For salt-sensitive applications, let the PE stand in the column for 2–5 min for better washing, and/or use two 500–750 µL washes. Sequential washes have the advantage of iterative dilution away of salts and may more reliably make cleaner DNA.
   • For best results, wash buffer can be aimed around the inside surface of the column to wash the surface earlier in contact with solubilization buffer, whose salts often contaminate gel-purified DNA, resulting in a 230 nm guanidinium absorption peak in spectrophotometry.
   • Keep wash flow-through away from the matrix and column walls by not jostling the collection tube after centrifugation. If the matrix gets contaminated through contact, add another wash step.
   • Instead of decanting flow-through from collection tube, aspirating it prevents its contaminants from spreading onto the side of the tube and later, the column and final elution tube.
   • When centrifuging, if >450 µL solubilization buffer was used, first aspirate out flow-through from collection tube into liquid waste, or else wash flow-through won't fit without contacting column matrix.
     
10. Dry the column by centrifuging 2 min to remove residual wash buffer.  (cannot be vacuumed) Meanwhile, you may preheat the eluent in a heat block.
    Then quickly remove from the centrifuge, discard the collection tube and place the column in a fresh, labeled 1.5 mL tube.
    (opt.) Allow the column to dry further for a minute at room temp or in the heat block.
    
    • Residual ethanol in matrix inhibits elution. Separate the dried columns from the collection tube right after centrifuging, or else elution is somewhat inhibited (by ethanol vapors recondensing on the matrix?) Allowing the 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.
11. Elute the DNA by adding 10–20 μL elution buffer EB or water (opt. heated) to the center of the micro column matrix.
    Let stand for 1–4 min, better in a heat block for large fragments.
    Centrifuge 30 s, ≥10000×g with tube caps braced for rotation.
    Check that column and tube labels match, and discard column.
    • You can elute with a larger volume, esp. for larger matrix/column, and yield may increase at the expense of DNA concentration. 
    • Warming your eluent, up to 60°C, before applying to the column can increase yield, especially for larger DNAs. Warm eluent to 42–60°C. QIAEX II recommendations:
      

      • Fragments <4 kb: Incubate at RT 5 min

      • Fragments 4–10 kb: Incubate at 50°C 5 min

      • Fragments >10 kb: Incubate at 50°C 10 min

    • Prefer an elution buffer to water; read notes in Materials.
      
    • Since the rotor turns counterclockwise, face elution tube caps clockwise over adjacent holes if there is space, or against the bottom of the adjacent tube, 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. This is why columns should also be labeled, so samples can still be identified.
    • Centrifuging at a lower speed than step 10 may prevent tube caps from breaking.

• Measure: DNA concentration and purity can be estimated using a NanoDrop™ spectrophotometer's microvolume pedestal using only 1–1.5 µL sample, or using a microplate reader, e.g., 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 very soon after generating and purifying, as the ends are not very stable for long periods even when frozen.