How to Recover More DNA from Agarose Gels

What Is Gel Electrophoresis?

Gel electrophoresis is a molecular biology technique utilized to analyze DNA and RNA size. Gel electrophoresis uses a charged electric field to pull negatively charged nucleic acid samples through a porous agarose gel matrix. The size of the nucleic acid fragment impacts the rate of migration, as smaller fragments have an easier time getting through the agarose matrix than larger fragments. For many workflows, gel electrophoresis is still the best way to separate specific sizes of DNA, especially for fragments larger than 1 kb.

Why Isolate DNA from Gels?

Following electrophoretic resolution, specific bands can be excised from the agarose gel matrix and processed to purify the DNA from the gel. Several applications, like cloning, still require resolving samples using agarose gels to separate out DNA fragments before performing any ligation reactions to ensure your final plasmid has the correct insert. However, recovering DNA from gel excisions can be challenging with many researchers struggling to recover more than 50% of their original input. Follow these tips for running your gel and extracting your bands to maximize the purity and yield of your recovered DNA.

Tips for Extracting DNA from Agarose Gels

• Know Before You Pour: Considerations for Running & Visualizing Your Gel

  • Compatibility is Key - It is important to validate the specifications of the kit to determine if it is compatible with the gel you need to extract from. For example, the Zymoclean Gel DNA Recovery Kits are compatible with both TAE & TBE buffered agarose gels.
  • Use Small Gel Wells - Additionally, we recommend choosing the combs that produce the smallest wells possible for the volume and amount of DNA to be resolved. This will concentrate the DNA into a smaller band, making it easier to excise as much DNA as possible while minimizing the amount of gel you need to dissolve.
  • Watch Your Percentages - It is good practice to consider the size of your DNA fragments when selecting an agarose gel percentage. A higher agarose gel percentage produces a tighter matrix gel and is typically used to visualize smaller fragments. Similarly, lower gel percentages may be needed to effectively visualize larger fragments. Most gel extraction kits can dissolve gels that contain up to 2 % agarose. However, if a gel is over 2% agarose, an additional volume of Agarose Dissolving Buffer is recommended to ensure the gel is completely dissolved prior to further processing.
  • Space Out the Bands - When loading the wells, it is good practice to load every other well, if possible, to give more space to cut around your bands of interest later. In addition, for the crispest bands run the gel at a lower voltage for a longer period of time.
  • Work Quickly - When visualizing the gel, it is important to minimize the time that the gel is exposed to UV light. The longer the sample is exposed to UV light the greater the risk of DNA damage. Therefore, it is highly recommended to work quickly and efficiently when imaging the gel and excising your bands of interest.

• Gel Excision: Cutting & Processing DNA Bands

  • Cut Close to the DNA Band - It is extremely important to ensure that the slice is cut as close to the desired DNA band as possible. Doing this reduces the amount of agarose that needs to be dissolved.
  • Gel Weight is Critical - Most gel extraction kits use a fixed ratio of agarose dissolving buffer (ADB) to gel weight. It is good practice to weigh the microcentrifuge tube first and then weigh again after you place the excised gel band in the tube to determine the exact weight of the gel. Most gel extraction kits can handle gel excisions up to 400 mg. If your gel excision weighs more than 400mg, the extraction will need to be scaled up accordingly or divided among multiple extractions.
  • Ensure Gel Slice Has Dissolved - It is critical to make sure that the agarose is completely dissolved before binding onto a column. If there are any traces of partially undissolved agarose, the gel could clog the column leading to an impure sample with a low 260/230 ratio. Moreover, undissolved agarose will retain DNA, decreasing your DNA yield. If you are unsure if the agarose gel is fully dissolved, vortex the ADB and gel excision solution. The mixture should be homogenous and relatively clear, without traces of gel pieces. If you’re not confident the gel is completely dissolved, incubate the solution for 10-15 minutes longer. There is no harm in a slightly longer incubation, but you will run into problems later if the gel is only partially dissolved.
  • Watch the Temperature - Although you can perform the dissolving step at room temperature, the gel will dissolve much faster at 55 °C. However, the temperature should not rise above 60°C as it increases the chances of denaturing the DNA, which will lower your recovery. When incubating your sample, it is best to use a water bath over a bead bath to maintain the correct temperature and uniform heating.
  • Elute Carefully - To ensure the best possible recovery, it is important that the eluted DNA is free of salts and other contaminants. To do this, be deliberate about the wash steps of the protocol. It is recommended to run the wash buffer along the edge of the column walls to flush out any of the salts that may be left over from the Agarose Dissolving Buffer. When eluting, be sure to place the elution buffer directly onto the column matrix. This will help to maximize your yield and prevent the eluate from collecting any salts that may have dried higher up on the column walls.

  • Keep Fragment Size in Consideration - Large DNA fragments (>11 kb) are tricky to recover as they are more difficult to elute from the column matrix. Standard kits, including the Zymoclean Gel DNA Recovery Kit, can recover up to 10 kb of DNA. For fragments in the upper range of this (8-10 kb), we recommend incubating the elution buffer on the column matrix for up to five minutes at room temperature. Additionally, using a pre-warmed (60-70 °C) elution buffer can help to increase the elution efficiency.

    For DNA fragments greater than 10 kb, we recommend using a kit validated on for high molecular weight DNA, such as the Zymoclean Large Fragment DNA Recovery Kit. This kit includes a column designed for easier elution of very large DNA, up to 200 kb.