Isolation of DNA from agarose gels for sequencing with the CEQ8000.
This procedure is based on (M.R. ISLAM, M. RODOVA, J. P. CALVET, 2002, A fast and efficient method of DNA fragment isolation from agarose gels without using commercial kits. American Biotechnology Laboratory 20:18).
1) Prepare fresh TAE buffer and a new agarose gel in TAE buffer. Use a clean electrophoresis chamber. Make the gel just thick enough to hold the DNA sample in a single well. Use the lowest concentration of agarose that will give the needed sample resolution.
2) Immediately following electrophoresis, transfer the gel and casting tray to a UV light source. Cut out the band of interest with a sterile spatula (flaming works well) and place it in a 1.5 mL tube. Crush the gel slice with the spatula.
3) Freeze at -80 C.
4) Thaw the sample at room temperature. Gently flick the tube to make sure that no ice remains. Centrifuge at full speed (20,000 x g or as close as you can get) for six minutes. Remove the supernatant (save) with a pipet tip to a fresh tube.
5) Crush the gel slice with the pipet tip.
6) Repeat steps 3 and 4 to recover more DNA from the gel slice.
7) Pool saved supernatants from steps 4 and 6 and centrifuge at full speed (20,000 x g or as close as you can get) for six minutes.
8) Remove the supernatant (save) with a pipet tip to a fresh tube.
9) If there is more than 0.4 mL of liquid, divide it into multiple tubes so there is a maximum of 0.4 mL per tube.
10) Add 1 uL of glycogen (20 mg/mL, Sigma G-1633, type VIII from slipper limpets) and 1/10th volume of 3 M acetate to each tube. Add 2 volumes of 95% ethanol, mix, and place at -20 C overnight.
11) Centrifuge at full speed (20,000 x g or as close as you can get) for fifteen minutes.
12) Decant the supernatant and invert the tube on a paper towel.
13) Add 0.5 mL of 70% ethanol and vortex. Centrifuge at full speed (20,000 x g or as close as you can get) for five minutes.
14) Decant the supernatant and invert the tube on a paper towel.
15) Dry the pellet in a speedvac.
16) Resuspend the pellet in 20 uL of water.