Isolating DNA fragment from agarose gel (provided by Chanhyo Lee, 6/4/03)

The following protocol is based on Song Tan's protocol.

1. Electrophorese the DNA sample on an agarose gel. While the gel is running, prepare the filter assembly as follows:

(1) Make a small hole at the bottom of an 0.5 ml Eppendorf tube with a hot needle.

(2) Pack some silanized glass wool (SUPELCO cat#. 20410 or 20411, www. Sigmaaldrich.com) in the bottom of the pierced Eppendorf tube.

(3) Place the tube into a labeled 1.5 ml Eppendorf tube.

2. Separation of DNA fragments can be visualized with long wavelength UV light from a hand-held lamp. Cut out the desired bands and place them in the 0.5 ml Eppendorf tube above the silanized glass wool.

3. Centrifuge the filter assembly at 7000 rpm for 5 minutes. Discard the 0.5 ml tube. The purified DNA fragment is in the 50 to 100 ul liquid in the 1.5ml tube. This gel purified DNA can be used in ligation reactions and PCR reactions without further processing.

Notes from Gilmour:

- Recovery of DNA is likely to be poor for DNA of several kilobases.

- Do not ethanol precipitate this DNA as the DNA will become trapped in impurities from the agarose gel. If you require further purification or concentration, try spermine precipitating the DNA.