Gel Extraction

When you have a heterogenous sample of DNA fragments of different lengths, you will do a gel extraction to separate the DNA pieces and purify the fragment(s) of interest. Follow gel electrophoresis guides when doing a gel extraction.

Protocol

In advance, weigh microcentrifuge tubes on the scale for as many DNA fragments you need to extract. This will be used to determine the weight of your extracted gel fragments, and the amount of dissolving buffer you will need to add. Turn on and set the heat block to 55°C, and add water to as many wells as you have tubes.

1. Make a 1% agarose gel using TAE. See TAE Gel under Media Recipes.

2. Add 5-10 µL of the appropriate ladder and DNA pre-mixed with loading dye (5 part DNA sample, 1 part loading dye) to the extra-wide wells in the agarose gel.

3. Run large gels between 90-120 V, and small gels between 60-90 V (running gels at too high of a current will cause the gel to melt/denature).

4. Verify that the gel holder is positioned with wells at negative terminus (black).

5. Verify hydrogen bubble production at negative terminus.

6. When DNA fragments are well separated, stop power supply.

7. Remove gel from container, and place gel directly on the blue-light transilluminator.

8. Identify the desired fragment. Use scalpel to cut an extremely thin piece of gel surrounding desired fragment. Minimize quantity of gel slice while maximizing DNA content. Transfer the gel fragment to your pre-weighed microcentrifuge tube.

9. Weigh the microcentrifuge tubes with the gel fragments.

10. Add 600-900 µL ADB (agarose dissolving buffer) according to how much your gel weighs, as described by the ADB container.

11. Transfer the closed tubes to the water-filled, preheated heat block.

12. Incubate for 20-30 minutes until the gel fragments are completely dissolved. Vortexing the tubes every 5-10 minutes helps accelerate this process.

13. Add solution to Zymo spin columns, place Zymo spin columns in collection tubes, centrifuge (spin) these at max speed for 30 seconds.

14. Add ~300 µL wash buffer, spin for another 30 sec.

15. Repeat step 14.

16. Spin again for 1 minute with no solutions added (a dry spin) at max RPM to remove all ethanol traces.

17. Transfer column to a new microcentrifuge tube and add 10-15 µL ddH2O water directly to the column matrix.

18. Incubate for 1 minute to maximize elution yield.

19. Spin at max speed for 2 minutes to elute.

20. Measure DNA concentration using the NanoDrop.

Tips and tricks

• Add your cut plasmid DNA to a single extra-wide well. If you split up your cut plasmid DNA across multiple wells, the DNA gel extraction step will have lower efficiency.

• Don’t be afraid of performing "surgery" on your gel slice. Slice off any portion which does not contain any DNA inside it. This will increase the extraction yield.

• For most gels, use the large combs (they have two sides).

• When making your gel, there is a chemical container labeled only as “agarose”. This is not what you want! Use the big container labeled “SeaKem LE Agarose”

• Remember that smaller fragments will travel faster than larger fragments.

• Take a picture of your gel once bands are sufficiently separated for future reference/dissemination.