Freeze-squeeze.htm

 

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.