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Purifying DNA from Gel Slices
From 2008.igem.org
Newcastle University
GOLD MEDAL WINNER 2008
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Home >> Wet Lab >> Protocols >> Purifying DNA from Gel Slices
To purify DNA from a slice of agarose gel we used the GE Healthcare illustra GFX PCR DNA and Gel Band Purification Kit, and the method outlined below is taken from their protocol manual included with the kit.
This can be found here:[http://www5.gelifesciences.com/aptrix/upp00919.nsf/content/EF527125F24048B9C12572BF0081201D?OpenDocument&Path=Catalog&Hometitle=Catalog&entry=1&newrel&LinkParent=C1256FC4003AED40-31B65D2972761A41C125701900490A45_RelatedLinksNew-57DF33EF62AB6478852573DE0066D351&newrel&hidesearchbox=yes&moduleid=39955]
1) Sample Capture
- Add 10 μl Capture buffer type 2 for each 10 mg of gel slice.
- Mix by inversion and incubate at 60°C until the agarose is completely dissolved. Mix by inversion every 3 minutes.
- Place a GFX MicroSpin column into a Collection tube.
2) Sample Binding
- Centrifuge Capture buffer type 2- sample mix briefly to collect the liquid at the bottom of the tube.
- Transfer 600 μl Capture buffer type 2- sample mix onto the assembled GFX MicroSpin column and Collection tube.
- Incubate at room temperature for 1 minute.
- Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
- Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin column back inside the Collection tube.
3) Wash & Dry
- Add 500 μl Wash buffer type 1 to the GFX MicroSpin column.
- Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
- Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube.
4) Elution
- Add 10–50 μl Elution buffer type 4 OR type 6 to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.
- Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.
- Spin the assembled column and sample Collection tube at 16 000 × g for 1 minute to recover the purified DNA.
- Store the purified DNA at -20°C.
