1. Excise band from gel using straight-edge razor blade. Weigh excised agarose piece to estimate its volume (1 g ~ 1 ml). Keep weight under 0.4 g if using 1.5-ml microcentrifuge tubes.

2. Add 3 volumes (1.2 ml for 0.4 g slice) NaI stock solution.

3. Incubate at 45°C to 55°C in water bath for 5 minutes, mixing after two minutes. Check to see if all agarose has melted after five minutes, if not, incubate a little longer.

4. Vortex glassmilk stock solution to mix well. Add 15-20 µl glassmilk to tube.

5. Incubate tube for 15 minutes on ice, mixing every 3 to 5 minutes to keep glassmilk in suspension.

6. Microcentrifuge tube on high for 5 seconds to pellet the glassmilk. Remove supernatant to new tube. Hold if critical.

7. Add 500 µl (10-50 volumes) ice-cold New Wash to silica gel pellet. Resuspend pellet.

8. Microfuge tube 10 seconds on high. Discard supernatant.

9. Repeat steps 7 and 8 two more times (3 washes total).

10. After discarding supernatant from last wash, spin pellet for 10 seconds, remove residual New Wash with pipette.

11. Resuspend pellet in 15 µl dH2O.

12. Incubate tube for 5 minutes at 45°C to 55°C in a water bath.

13. Microfuge tube on high for 30 seconds to pellet silica gel.

14. Transfer DNA-containing supernatant to new tube.

15. Repeat steps 11 through 14, combining supernatants.

16. Spin the tube containing supernatants collected from step 15 for a few seconds on high to remove any residual glassmilk carried over with supernatant transfer. Transfer supernatant to new tube.

Notes on GeneClean II Procedure:

1. A tube capable of holding 3 to 4 times the volume of the gel slice (1 g ~ 1 ml) will be required. Large slices of gel can be sliced into ~2 mm cubes to aid gel dissolution during next step.

2. Keep final concentration of NaI above 4 M. For gels containing TBE, add 0.5 volume TBE modifier and 4.5 volumes of NaI to a given volume of agarose.

3. If DNA is not contained in agarose, skip water bath incubation step.

4. It may take some time (2-3 minutes) of vortexing to adequately re-mix glassmilk. Resuspending glassmilk is easier if you hold tube sideways when vortexing, and store tube on its side when not in use.

5. This quantity is empirically derived. The binding capacity of the silica matrix is ~1 µg to 2 µg DNA per 1 µl of suspension. The purpose is to provide a surface area that is in large excess over that required by all DNA in solution to speed binding. Rule of thumb: Add 5 µl GM suspensions to solutions containing 5 µg or less DNA, add an additional 1 µl GM for each 0.5 µg DNA.

6. This incubation may also be at room temperature. Instructions with the kit specify 5 minutes at room temperature, but recovery appears greater if done on ice for a longer period of time. If the volume of the binding reaction exceeds 1.5 ml, allow a longer incubation.

7. At this point, the DNA is presumable adhered to the silica beads (glassmilk) pelleted in step 8. Care should be exercised with the supernatant at this point in case adhesion did not occur efficiently.

8. The GM pellet is somewhat difficult to resuspend. Use pipette tip and pipette back and forth to aid in resuspension.

9. Resuspend the pellet in an amount of dH2O or low-salt buffer equal to the amount of glassmilk originally added.

10. This second elution step is optional. The manufacturer claims that ~80% of the bound DNA is recovered with the first elution. A second elution may result in a 10-20% additional recovery of bound DNA.

11. This last step removes trace amounts of silica gel carried over from the elution. The silica gel will not interfere with subsequent use of the DNA, and DNA will not bind to it in less than 3 M salt. If this step is skipped, spin DNA for 10 seconds prior to using.