Isolation of DNA from Agarose Gels (Paper Slurry Method)
This procedure isolates DNA from agarose gels by filtration through a filter-paper column. The column is made in a 500 µL tube from a slurry of filter paper in TE buffer.
- Whatman 3MM filter paper: 50 cm2 piece
- TE Buffer (10X): dissolve 186 mg EDTA and 605 mg Tris in 50 mL dH2O; pH to 8.0 with HCl. Store at 4°C, dilute ten times before using.
- Paper Slurry: cut 50 cm2 of filter paper into tiny (1-2 mm2) pieces; add to 40 mL of TE buffer in a 50 mL tube. Shake vigorously by hand for at least 5 minutes. Store at 4°C.
- Agarose gel with DNA to be isolated
- Clean razor blade
- Filter column: Punch a small hole in the bottom of a 500 µL tube with a 23 gauge needle. Remove paper slurry piecewise with tweezers and place over the hole in the 500 µL tube. Pack with a 200 µL pipet tip and remove excess liquid. Repeat until paper column is approximately 3 mm high. Place inside a 1.5 mL tube to collect eluent.
- Excise the DNA band from the surrounding gel with a clean razor blade; be sure to remove as little gel as possible.
- Dice the excised gel fragment into small pieces with the same razor; transfer onto filter column.
- Centrifuge for 10 minutes at highest speed (approximately 20,000g) in a microcentrifuge.
- Transfer eluent to a fresh tube; recentrifuge agarose
- Combine eluent with that from previous centrifugation
- Assemble a second spin column and transfer remaining agarose to the new column. Spin again.
- Combine all eluent fractions and concentrate via cold ethanol precipitation
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