Page History

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 get the obtain desired amount of a particular NADNA.
  • Label the tubes with sample names. One will be used for gel melting and one for the product.
• DNA or RNA purification column and collection tube. Often low, 5 µg-capacity to allow smaller elution column (~10–15 µg capacity) to allow high concentration.volume and thus higher DNA concentration.
  • Zymo-I column: 5 µg DNA/RNA, ≥6 µL eluate, 800 µL capacity
  • Econospin: 30–40 µg DNA, ≥10 µL eluate, 800 µL capacity
• Collection tube, used to collect column flow-through waste.
• Qiagen Buffer QX1, an agarose Agarose dissolving buffer (≥ 3 µL/µg mg gel fragment)
  • Can substitute Qiagen Buffer QG or Zymo Agarose-Dissolving Buffer, with modified disposal and optimization protocols. See next section for comparison.
• Qiagen Buffer PE, column Column wash buffer (≥ 400 µL/sample). Ensure ethanol was added.
• Eluent (elution buffer/TE or nuclease-free water), optionally heated to ≤65°C
  • Elution buffer elutes better because of buffering to elution pH and better DNA solvation than water, and it protects DNA from degradation with buffered alkalinity, especially across freeze-thaws when drastic local pH changes are damaging.
• Gel viewing equipment
  • Blue light transilluminator, orange shield/glasses for blue/green dyes (GelGreen)
  • UV transilluminator, 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.

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 NaPO4, 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 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.
• The Bennett Lab has phased in QX1 as our standard, replacing Qiagen QG and Zymo ADB/wash buffers, which are to be collected separately if used, so as to not be bleached with the majority of column purification 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 750 µL wash as in a miniprep 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 inside the silica pellet and thus require more and larger washes.

Procedure

Summary: Cut bands into tubes. Add ≈400 µL ADB. Melt for 10 min~400 µL or 3 volumes QX1 and dissolve ~10 min, 50°. Pass through column. Wash column twice with 200 µL wash bufferPE. Spin dry 2 min. Elute in ~10 µL. 

1. Visualize nucleic acids DNA on gel placed on tray/dish over transilluminationblue transilluminator.
   Capture image for later reference, as excision will destroy the gel.
2. (opt.) To be surer about complete melting, weigh each tube and record massesTare the analytical balance with an empty 1.5 mL tube.
3. 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. If desired band is close to undesired ones, be more conservative in cut placement to reduce capture of undesired NAsundesired DNAs.
   • Use the smallest agarose fragment possible. The less agarose in solution, the more efficient the extraction will be. Larger fragments take longer to melt and require more dissolving buffer, which introduces more salts, etc to the purification. Dissolved fragments >200 mg will not fit with 3× dissolving buffer in the columns.
4. (opt.) Reweigh tubes to calculate for gel fragment massmasses.
5. MeltDissolve: Add 300–400 µL agarose dissolving buffer (or more exactly 3 µL/µg gel) 3 µL Buffer QX1 (or QG or ADB) per milligram gel to each sample and ensure gel fragments are in buffer, or else centrifuge or knock /push them down into it. Salt concentration impacts DNA fragment affinity to silica according to size:
   • 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 42–55°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.
   • The solubilized gel color should be yellow, indicating low enough pH for silica binding. If orange or purple, add 10 µL 3 M NaAc pH 5 (in a tube in the cabinet).
6. (opt.) Depending on kitIf using Buffer QG or ADB, add 1 µL isopropanol/µg mg gel for higher yields of DNA fragments <500 bp and >4 kb. Vortex to mix.
7. (opt.) Cool: Allow samples to cool a few minutes min to room temp to enhance NA DNA binding to silica
8. Bind: Pipette or pour dissolved agarose samples into low-capacity columns to bind NAs bind DNAs to matrix.
   Centrifuge ≥18,000 rcf, 30 s. Or apply sample to column fit into vacuum manifold (faster) and apply vacuum until liquid is passed throughthrough (requires strong vacuum pressure).
9. Wash2×Wash: Add 200 µL wash buffer PE to column, and similarly centrifuge or apply vacuum. Repeat with a second 200 µL wash. Empty the collection tube.
   • If >450 µL agarose dissolving buffer was used, first pipette or aspirate out flow-through from collection tube into liquid waste, or else wash flow-through won't fit without contacting column matrix.
   • Keep flow-through away from the matrix and column walls by not jostling the collection tube.
   Wash: Again add 200 µL wash buffer to column, and similarly centrifuge or apply vacuum
   • Aspiration instead of decanting of collection tube contents prevents flow-through salts from spreading onto the side of the column and contaminating the final elution tube.
     
10. Dry: Empty the collection tube and centrifuge Centrifuge column 2 min with emptied collection tube to dry it from ethanol. (Meanwhile, preheat eluent.)
    Then 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. Ethanol vapor from collection tube was found to inhibit elution, thus the quick removal of the collection tube after centrifuging to dry.
      
    • Optionally, let the column in the elution tube sit at room temp or in a 42–60°C block or bead bath for a few minutes to further assist drying of ethanol, potentially improving yield. In the next step, you can add preheated eluent to the heated column and let incubate a few more minutes before centrifuging. Heat the eluent only before use to preserve elution buffer and plastic integrity.
11. Elute: Add Elute: Place the column into remaining clean 1.5 mL tube. Add 10–20 µL eluent (elution buffer or water), optionally heated. Wait 1–4 min before centrifuging ~18centrifuging ≥15,000 rcf, 30 s. Discard column.
    • Heating your eluent before applying to the column can increase yield, especially for large fragments (≥10 kb).Warm eluent to 50–65°C.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

    • Elution buffer (TE or Tris-HCl pH 8) elutes most effectively Elution buffer elutes better because of buffering to optimal elution pH and better DNA solvation than water, and it protects DNA from degradation with buffered alkalinity, especially across freeze-thaws when drastic local pH changes are damaging.can be damaging. DNA to be electroporated in larger quantity is better eluted in water to minimize salt/ion concentration in the eluted DNA to keep high electrical resistance.

• DNA NA concentration and purity can be estimated using a NanoDrop™ spectrophotometer's microvolume pedestal without sacrificing but 11–1.5–2 5 µL sample. It can show presence of high levels of typical salt contaminants (especially guanidinium) via the 260/230 nm absorbance ratio.
• Store purified NAs at 20°Cpurified 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 /4°C during usageor 1°C CoolSafe during usage. Linear DNA is more prone to degradation at ends than circular plasmid DNA.