Getting ready to perform a gel extraction?Here are six tips to help you get the best results possible.
- Melt your agarose completely
The number one reason that users see low yields with gel extraction procedures is because the agarose plug is not completely melted. When this happens, DNA remains trapped inside the agarose and cannot be extracted properly.
- Minimize exposure to UV light
Excise the gel slice as quickly as possible, as exposure to UV light damages DNA. As long as the excision is done quickly, damage done to the DNA will be negligible.
- Use the smallest agarose plug possible
There are many reasons for this one! The less agarose in solution, the more efficient the extraction will be. The larger your agarose plug is, the longer it will take to melt. It will also require more dissolving buffer, which introduces more salts and other components to the purification. Also, if the plug is greater than 160 mg, the volume of agarose plus buffer will exceed the volume of the column reservoir (800 µl), and will require that your sample be loaded onto the column in two steps.
- Ensure there is no ethanol in your eluate
Ethanol contamination can interfere with downstream applications. Frits, which are common in many manufacturers’ columns, often retain small droplets of buffer, also known as buffer retention. This can contaminate your eluate. Monarch® columns are designed without a frit, which eliminates buffer retention.
If you suspect that the tip of your Monarch column has come into contact with wash buffer after a spin, give the column another short spin to ensure that your column is ethanol-free.
- Warm your Elution Buffer to 50°C for large fragments
Heating your Elution Buffer before applying to the column can increase efficiency, especially for large fragments (>10 kb).
- Use the recommended amount of Dissolving Buffer
The Monarch protocol requires four volumes of dissolving buffer to be added to your gel slice. Other manufacturers’ protocols differ, so when using our kit, be sure to use the recommended volume.