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PCR or restriction enzyme digestion sometimes results in a mixture of DNA products, although you only want one for your next step. To isolate the piece of DNA you want, you can separate the DNA products by agarose gel electrophoresis, visualize the DNA bands, and then you can cut out the piece of the gel containing only your desired piece of DNA. Gel extraction can then be performed to remove the DNA from the gel. Like the PCR cleanup, this will also purify the DNA from any buffer components that are incompatible with downstream procedures. We will use a kit to perform the gel extraction protocol.
- 1.5ml microcentrifuge tubes
- Razor or scalpel
- Gel Extraction Kit
Centrifugation should be performed at approx. 10,000g unless otherwise specified.
Note: Instructions provided are for the Zymo agarose gel extraction kit. While all gel extraction kits utilize similar protocols, there may be small but significant differences between manufacturers. If using another kit, please follow the instructions listed by the manufacturer for steps 4-9.
- Weigh an empty 1.5mL microcentrifuge tube and record its mass.
- Excise the DNA fragment from the agarose gel using a razor blade or scalpel and transfer it into a 1.5 ml microcentrifuge tube. Cut away as much unnecessary agarose as possible.
- Weigh the tube containing the agarose gel slice again to determine the mass of the gel slice in the tube.
- Add 3 volumes of Zymo ADB to each volume of agarose excised from the gel (e.g. for 100 mg of agarose gel slice add 300 µl of ADB)
- Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved. For DNA fragments > 8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g., 100 µl agarose, 300 µl ADB, and 100 µl water).
- Transfer the melted agarose solution to a Zymo-Spin Column in a Collection Tube.
- Centrifuge for 30 seconds. Discard the flow-through.
- Add 200 µl of Zymo DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through. Repeat the wash step. Spin without wash for 1 minute at maximum speed to remove any residual wash buffer.
- Add 10 µl water directly to the column matrix and incubate at room temperature for one minute. Place the column into a 1.5 ml tube labeled as “GFP insert” and centrifuge for 30 seconds to elute DNA. Ultra-pure DNA is now ready for use.
- Quantify the DNA concentration using the Nanodrop. Record the concentration of DNA on the side of your tube.
Store DNA at 4C until ready to use.