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 get the desired amount of a particular NA.
  • 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 elution column (~10–15 µg capacity) to allow high concentration.
• Agarose dissolving buffer (≥ 3 µL/µg gel fragment)
• 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.

Procedure

Summary: Cut bands into tubes. Add ≈400 µL ADB. Melt for 10 min. Pass through column. Wash column twice with 200 µL wash buffer. Spin dry 2 min. 

1. Visualize nucleic acids on gel placed on tray/dish over transillumination.
   Capture image for later reference, as excision will destroy the gel.
2. (opt.) To be surer about complete melting, weigh each tube and record masses.
3. Excise desired bands precisely with blade and scrape into 1.5 mL tube. Minimize contact with other bands. If desired band is close to undesired ones, be more conservative in cut placement to reduce capture of undesired NAs.
   • 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 gel fragment mass.
5. Melt: Add 300–400 µL agarose dissolving buffer (or more exactly 3 µL/µg gel) to each sample and ensure gel fragments are in buffer, or else centrifuge or knock/push them down into it.
   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.
6. (opt.) Depending on kit, add 1 µL isopropanol/µg gel for higher yields of DNA fragments <500 bp and >4 kb.
7. (opt.) Cool: Allow samples to cool a few minutes to enhance NA binding to silica
8. Bind: Pipette or pour dissolved agarose samples into columns to bind NAs 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 through.
9. Wash: Add 200 µL wash buffer to column, and similarly centrifuge or apply vacuum.
   • 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.
10. Wash: Again add 200 µL wash buffer to column, and similarly centrifuge or apply vacuum.
11. Dry: Empty the collection tube and centrifuge column 2 min to dry it from ethanol.
    • Residual ethanol inhibits elution.
12. 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 ~18,000 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.
    • 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.

• NA concentration and purity can be estimated using a NanoDrop™ spectrophotometer without sacrificing but 1.5–2 µL sample. It can show presence of typical contaminants.
• Store purified NAs at 20°C. Thaw at room temp and (for best practice) keep on ice/4°C during usage.