A gel extraction is used to select a fragment of DNA of a specific length out of a solution composed of different fragments (ideally the difference in length between the wanted fragment and the closest-sized fragment should be more than 200bp). These fragments are often obtained after a digestion.

The yeild for this procedure is typically very poor so a large amount of starting material, digested DNA in this case is required. We typically used 4 micrograms. The digestion products are loaded on a gel. Lanes on both sides of the one to be extracted should be empty to make cutting easier and avoid contamination with other fragments.

The gel should be run long enough for the bands to be spread out. This is particularly important if the fragment of interest is around the same length as other expected digestion products. UV light is necesary to observe the bands on the gel but exposure time should be minimized to avoid DNA damage. The fragment of interest is then excise and put in an Eppendorf (consider using a 2ml one).

To extract the DNA from the agarose we used Macherey-Nagel's "Nucleospin® Gel and PCR clean-up" kit. Its manual can be found here: Gel and PCR clean-up Manual

Tips

• Cut away as much Agar as possible without slicing into the DNA. Excess agar will require more solvent to dissolve and will result in a poorer yeild upon elution.
• Minimize the DNA's exposure to the UV-light. UV will damage DNA and have negative effects on any subsequent reactions (for example, ligations can be 10'000x less effective when DNA has been exposed to too much UV light [1]