GENECLEAN GLASSMILK PROCEDURE

1. Excise DNA band from EtBr stained gel. Determine volume by weight. Use 1.5 ml tube if weight less than 0.4 g. Volume will be 3-4 times the weight of the gel slice. 2 mm cubes will facilitate gel dissolution.
2. Add at least 2, but not more than 3 volumes NaI stock solution. The concentration of NaI will be close to 4 M. If agarose is present, place tube in a 45-55C water bath and mix just long enough to dissolve the agarose.
3. Add Glassmilk. Vortex Glassmilk stock until all contents are in suspension. This requires about one minute of vigorous shaking. Add 5 ul of Glassmilk suspension to solutions containing 5 ug or less and add an additional 1 ul for each 5 ug of DNA above 5 ug. Mix and place on ice (or RT) for 5 min to allow binding of the DNA. It is not necessary to continue incubation longer than 5 min.
4. Pellet silica matrix with the bound DNA. Spin about 5 sec for 1.5 ml microfuge tubes. Spin 1-2 min for larger tubes then in step 5 transfer the resuspended pellet to a 1.5 ml microfuge tube. Remove and discard supernatant with pipet or pour (saving supernatant is optional if you suspect that all the DNA was not bound by Glassmilk.). If agarose is still present, add 200-400 ul 1/3 diluted NaI stock and dissolve agarose in 45-55C water bath for a few minutes. Re-pellet matrix and discard supernatant.
5. Wash pellet 3 times with NEW. Add approximately 10-50 volumes (approx. 200-700 ul is convenient) ice-cold NEW. Resuspend the pellet by trituration. Spin 1.5 ml tube for 5 sec. and discard the supernatant. Repeat the wash procedure 2 more times. After the final wash re-pellet the matrix and remove as much of the supernatant as possible. In this process, DNA fragments greater than 15 kb are likely to be sheared. Try to be gentle during the pellet resuspension steps if your DNA fragments are large.
6. Elute DNA from glassmilk. Resuspend washed, white pellet with TE buffer, water, or low salt buffer of choice. The pellet easily resuspends in a buffer volume equal to the volume of the pellet (5 ul or more buffer if 10 ul Glassmilk used.). Incubate the tube in a 45-55C waterbath for 2-3 minutes. Centrifuge about 30 sec to make a solid pellet. Carefully remove the supernatant containing the eluted DNA. A second elution from the white pellet may recover 10-20% additional DNA.

Yields should be greater than 80%

More Information:

• Hamaguchi, K., and Geiduschek, E.P. 1962. J.Amer.Chem.Soc. 84:1329.
• Struhl, K. 1985. BioTechniques 3:452.
• Vogelstein, B., and Gillespie, D. 1979. Proc.Natl.Acad.Sci.USA 76:615.