DNA extraction from Agarose gel

Goal

Protocol

1. Excise DNA fragment with a scalpel.


2. Add 200 µL Buffer NT for each 100 mg agarose gel. Incubate sample at 50°C until the gel slices are dissolved.



SAFETY AND USEFUL RECOMMANDATION
The Membrane Binding Solution is considered as harmful that is why in case of liquid on the skin or in the eyes, it must be washed with soap and water. However the quantity is small therefore the risk is minimum. The gel is also full of ethidium bromide. This product is not volatile, and its melting point is around 240∞C, therefore the inhalation risk is minimal.



3. Place a NucleoSpin® Extract II column into a 2 mL collecting tube and load the sample. Centrifuge for 1 min at 11,000 x g.



SAFETY AND USEFUL RECOMMANDATION
The centrifuge must be balanced before being switched on. A dysfunctioning device must not be used.



4. Discard flow-through and place the column back into the collecting tube.


5. Add 600-700 µL buffer NT3. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the column back into the collecting tube.



SAFETY AND USEFUL RECOMMANDATION
The membrane wash solution contains ethanol. When using the centrifuge, the lid is closed.Leading to a possible accumulation of ethanol gas. It is thus important to open the lid once manipulations are finished to evacuate the gas.



6. Centrifuge for 2 min at 11,000 x g. You can also incubate the column for 2-5 min at 70°C prior to elution.


7. Place the column into a new clean 1.5 mL eppendorf tube. Add 15-50 µL elution buffer NE, incubate at room temperature for 1 min. Centrifuge for 1 min at 11,000 x g.