Obtaining DNA from Agarose Gels (Paper Slurry Method) 凝胶中回收DNA【University

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.

Materials

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℃, 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℃.

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.

Procedure

1.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 agarose

5.Combine eluent with that from previous centrifugation

6.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