Isolation of DNA from Agarose Gels(Paper Slurry Method)

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.

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