Phenol Chloroform Gel Extraction Protocol

This protocol is useful for extracting DNA from an agarose gel after electrophoresis. It has not been optimized, but is generally successful.

1. Cut the band you want to extract DNA from using a single-use razor blade while the gel is in the UV box. Make sure to have the UV light setting on low so you don't start to harm the DNA. Exposure to large amounts of UV light can damage your DNA.

2. In a small petri dish, use the razor blade to chop the piece of gel into small pieces. Transfer to a microcentrifuge tube and crush with a P1000 pipet tip.

3. Add 1 volume of phenol and vortex vigorously.

4. Freeze at -80 degrees Celsius for 30 minutes.

5. Centrifuge for 10 minutes at 13,000 rpm. The DNA will be in the top layer.

6. Move the top layer to a new microcentrifuge tube and add 1 volume of phenol. Vortex it to mix.

7. Spin again.

8. Remove the top layer again and place it in a new tube.

9. Make a 1:1 mixture of phenol and chloroform, making sure to vortex prior to pipetting into the DNA solution. Add 1 volume of this mixture to each tube of DNA. Vortex to mix.

10. Centrifuge again.

11. Remove the top layer and add it to a new tube. You may want to use a larger tube for the following steps, as the volume increases substantially.

12. Add 1:10 volume of 3M NaOAc and vortex.

13. Add 2.5 volumes EtOH (on ice).

14. Let this mixture chill at -20 degrees Celsius for 20 minutes. (Be very careful when handling the tubes at this stage.)

15. Centrifuge for 30 minutes.

16. Remove the liquid carefully, as to avoid disturbing the pellet. It contains your DNA.

17. Let the tube dry for 5-7 minutes at 65 degrees Celsius.

18. Add desired amount of Elution Buffer, usually 30-50 uL. Wash the walls of the tube with the added buffer to ensure dried DNA is in solution. This should also mix the solution well.

19. Let the solution sit at room temperature for five minutes.

20. Measure the concentration.