琼脂糖凝胶回收DNA――纸浆法

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.Materials1. Whatman 3MM filter paper: 50 cm2 piece2. 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.3. 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.4. Agarose gel with DNA to be isolated5. Clean razor blade6. 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.Procedure1. Excise the DNA band from the surrounding gel with a clean razor blade; be sure to remove as little gel as possible.2. Dice the excised gel fragment into small pieces with the same razor; transfer onto filter column.3. Centrifuge for 10 minutes at highest speed (approximately 20,000g) in a microcentrifuge.4. Transfer eluent to a fresh tube; recentrifuge agarose5. Combine eluent with that from previous centrifugation6. Assemble a second spin column and transfer remaining agarose to the new column. Spin again.7. Combine all eluent fractions and concentrate via cold ethanol precipitation