Technical
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Detailed Protocol: (Explains what is occurring at each step)
To isolate DNA from an Agarose gel
1. Cut desired DNA band from a TAE or TBE agarose gel.
What’s happening: The agarose gel slice containing the desired DNA is removed from the rest of the gel.
2. Determine gel band weight. This can be done in a spin filter or separate tube.
3. The following procedures should be carried out directly in the spin filter (for up to 0.2 gm of gel).
For > 0.2 gm or to process in a separate tube, see Hints page for procedure.
4. Place gel in the spin filter basket. Be sure gel is resting on the white filter membrane. The maximum
capacity per spin filter is 0.2 gm.
What’s happening: By placing the gel directly in the spin filter you can save tubes and time. The gel will
be melted in the spin filter.
5. Add 3 volumes of GelBind to the gel slice. For example, 0.1 gm agarose requires 0.3 ml (300µl) of
GelBind. Be sure gel is submerged in the GelBind buffer. Close lid.
6. Incubate for 2 minutes at 55°C. Invert once. Incubate one minute more or until gel is melted. (> 2% gels
can require more than 5 minutes to melt).
What’s happening: The gel should melt within a few minutes or longer depending on the agarose
percentage. Gels above 1.2% can take up to 5 minutes to melt. At this point the gel should be irreversibly
melted. It will not re-solidify at room temperature.
7. Invert once to mix.
What’s happening: The mixing step is important to keep the proper salt concentration.
8. Centrifuge spin filter 10 seconds at 10,000 x g.
What’s happening: The melted gel and unwanted salts flow through the spin filter membrane as the DNA
binds to the membrane.
9. Very important. Remove the spin filter. Vortex the collection tube for 5 seconds to mix the flow through.
What’s happening: When the gel melted, the DNA entered a salt solution. The gel however, does not
melt evenly. In most cases all the DNA does not bind on the first pass through the spin filter. The mixing
step makes the salt concentration homogeneous.
10. Reload all the liquid from collection tube back onto the spin filter.
What’s happening: Passing the DNA through the spin filter a second time will now allow any unbound
DNA to bind to the spin filter. This greatly increases the yield of DNA from the melted agarose.
11. Centrifuge 10 seconds at 10,000 x g.
12. Discard flow through liquid and replace spin filter basket.
What’s happening: All available DNA should be bound to the spin filter membrane. The discarded liquid
contains only melted agarose and salt.
13. Add 300 µl GelWash buffer.
What’s happening: GelWash is composed of an ethanol solution. It keeps the DNA bound to the spin
filter while washing away residual salt and melted agarose.
14. Centrifuge 10 seconds at 10,000 x g.
15. Discard flow through and centrifuge again for 30 seconds at 10,000 x g.
What’s happening: It is important to dry out any residual GelWash because the ethanol contained in this
buffer can inhibit down stream applications for the recovered DNA.
16. Carefully transfer filter basket to a clean collection tube provided.
17. Add 50µl of Elution buffer (10 mM Tris) or water directly onto the center of the white spin filter
membrane.
What’s happening: The elution buffer must contact the entire surface of the spin filter membrane for
efficient recovery of the DNA. The DNA is released from the spin filter membrane at this point because
there is no salt to keep it bound.
18. Centrifuge 30 seconds at 10,000 x g. Discard filter basket.
What’s happening: The DNA flows through the spin filter membrane and into the collection tube.
19. DNA is now ready to use. Thank you for choosing the UltraClean GelSpin Kit.
