Gel electrophoresis is used to identify, quantify, and purify nucleic acids. Once nucleic acids have been separated on an agarose gel, desired nucleic acid bands can be excised and purified away from the gel by melting the gel fragment and selectively binding its nucleic acids to a silica column, from which they are eluted.
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
- Elution buffer
- 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.
- Properly visualize nucleic acids on gel placed on tray/dish over transillumination.
Capture image for later reference, as excision will destroy the gel. - (opt.) To be surer about complete melting, weigh each tube and record masses.
- 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.
- (opt.) Reweigh tubes to calculate gel fragment mass.
- 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. - (opt.) Depending on kit, add 1 µL isopropanol/µg gel for higher yields of DNA fragments <500 bp and >4 kb.
- (opt.) Cool: Allow samples to cool a few minutes to enhance NA binding to silica
- Bind: Pipette or pour molten gel samples into column to bind NAs to matrix.
Centrifuge ~18,000 rcf, 30 s. Or apply sample to column fit into vacuum manifold and apply vacuum until flown through. - 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.
- Wash: Again add 200 µL wash buffer to column and similarly centrifuge or apply vacuum.
- Dry: Empty the collection tube and centrifuge column 2 min to dry it from ethanol.
- Elute: Place the column into remaining clean 1.5 mL tube. Add 10–20 µL eluent, optionally heated. Wait 1–4 min before centrifuging ~18,000 rcf, 30 s. Discard column.
- 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.