Maheshri:GelEx

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Protocol for the QIAEX II Gel Extraction Kit

This protocol is designed for the extraction of 40 bp to 50 kb DNA fragments from 0.3–2% standard or low-melt agarose gels in TAE or TBE buffers.

Important points before starting

• The yellow color of Buffer QX1 indicates a pH <=7.5.
• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• A heating block or water bath at 50°C is required.
• 3 M sodium acetate, pH 5.0, may be necessary.
• All centrifugation steps are at maximum speed (≥10,000 x g, ~13,000 rpm) in a conventional, table-top microcentrifuge.
• Mix by gently flicking the tube to avoid shearing the DNA. Do not vortex the tube.

Procedure

1. Excise the DNA band from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing excess agarose. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QX1 to 1 volume of gel for DNA fragments 100 bp – 4 kb. For example, add 300 μl of Buffer QX1 to each 100 mg of gel. For larger or smaller DNA fragments:
   • DNA fragments <100 bp - Add 6 volumes of Buffer QX1
   • DNA fragments >4 kb - Add 3 volumes of Buffer QX1 plus 2 volumes of H2O
   • >2% or Metaphor agarose gels - Add 6 volumes of Buffer QX1
3. Resuspend QIAEX II by vortexing for 10 s. Add QIAEX II to the sample according to the following.
   • <2 μg DNA - Add 10 μl of QIAEX II
   • 2–10 μg DNA - Add 30 μl of QIAEX II
   • Each additional 10 μg DNA - Add additional 30 μl of QIAEX II
4. Incubate at 50°C for 10 min to solubilize the agarose and bind the DNA. Mix by inverting the tubes every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow. If the color of the mixture is orange or purple, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color should turn to yellow. The incubation should then be continued for an additional 5 min at least. The adsorption of DNA to QIAEX II particles is only efficient at pH <=7.5. Buffer QX1 now contains a pH indicator which is yellow at pH <=7.5, and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Centrifuge the sample by quick spin and dump the supernatant.
6. Wash the pellet with 500 μl of Buffer QX1. Resuspend the pellet by shaking the tube. Centrifuge the sample by quick spin and remove all supernatant. This wash step removes residual agarose contaminants.
7. Wash the pellet twice with 500 μl of Buffer PE. Resuspend the pellet by shaking the tube. Centrifuge the sample by quick spin and dump the supernatant with a pipet. These washing steps remove residual salt contaminants.
8. Dry the pellet for 10-15 min in a 37°C incubator until the pellet becomes white. If 30 μl of QIAEX II suspension is used, dry the pellet for approximately 30 min.
9. To elute DNA, add 20 μl of ddH2O pre-warmed to 50°C and resuspend the pellet by pipetting. Incubate the suspension at 50°C for 5 min. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5.
10. Centrifuge for 30 s. Carefully pipet the supernatant into a clean tube. The supernatant now contains the purified DNA.

Final Note:

• Store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) but the EDTA may inhibit subsequent enzymatic reactions.