Hi, my name is Lisa Meduzia. I am a research scientist here at NEB.
Today I'm going to tell you how to use the Monarch DNA Gel Extraction Kit. Before you get started, add 4 volumes of ethanol to one volume of DNA Wash Buffer according to the instructions on the bottle’s label. All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM)
The first step of the protocol is to excise the gel fragment. Visualize the DNA fragment of interest under UV light, and excise it from the gel using a razor blade, scalpel, or other tool. Try to cut the agarose as close to the DNA fragment as possible to avoid excising excess agarose, and limit UV exposure as much as possible.
Next, dissolve or melt the agarose. Transfer the excised band to a 1.5 ml microcentrifuge tube and weigh the gel slice. Add 4 volumes of Monarch Gel Dissolving Buffer to the tube. For example, add 400 μl of Gel Dissolving Buffer to 100 mg or 100 μl of gel. Incubate the sample between 37-55°C for 5-10 minutes to melt the agarose, mixing periodically to ensure that the agarose completely dissolves. Incomplete dissolving will result in lower yields.
Next, bind the DNA. Once the gel slice has dissolved, load the sample onto the column and close the cap. Centrifuge the sample for 1 minute and discard the flow-through. The DNA is now bound to the column's matrix.
Now, wash the DNA. Re-insert the column into a collection tube and add 200 μl of DNA Wash Buffer. Centrifuge for 1 minute. You can discard the flow-through, but it is optional. Repeat the wash step by adding 200 μl of DNA Wash Buffer and spin for 1 minute.
In the final step, elute the DNA. Transfer the column to a clean microfuge tube, making sure that the tip of the column does not come into contact with the flow-through. Add 6 or more μl of DNA Elution Buffer to the center of the column membrane. Incubate for 1 minute. This incubation step is important for maximizing yields. Centrifuge for 1 minute and collect the flow-through, which contains your gel-extracted DNA.
For more information, please refer to the product manual found on our web site.
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