Dominik Haudenschild, February 7, 2001, from BioRad Protocol

1) Cut DNA from ethidium-bromide stained agarose gel. Weigh, assume 1g=1ml.

2) Add 3 volumes of Prep-a-Gene binding buffer to gel slice, then heat the tube at 37°C to 56°C until agarose is dissolved.

3) Add enough Prep-a-Gene matrix to bind all of the DNA present. Capacity is 0.2µg supercoiled DNA per microliter of matrix. Incubate 5 to 10 minutes at RT

4) Pellet Prep-a-Gene matrix (which now has DNA bound to it) by centrifuging about 30 seconds. Remove supernatant, and rinse pellet twice by resuspending it in 50 pellet volumes of binding buffer.

5) Wash pellet three times using 50 pellet volumes of Wash buffer.

6) Remove all remaining traces of wash buffer by spinning pellet dry, then removing all liquid with pipette tip.

7) Elute DNA with 1 pellet volume of TE. About 75% recovery of DNA is in this first elution. Elute again with another pellet volume of TE, to get an additional 10 to 15% DNA recovered. All Ethidium bromide should be removed, and the DNA can be used for sequencing, ligation, and restriction enzyme digestion.

Wash Buffer
40mM Tris, 4mM EDTA, 0.8M NaCl, pH 7.4. Add 1 Volume 95% ethanol before use.

Elution Buffer
10mM Tris, 1mM EDTA, pH 8.0