• Removal and purification of DNA from agarose gels:

GENECLEAN^® can separate DNA from agarose based on the binding properties of DNA to silica.
 
• Substitution for RNase and the phenol/chloroform extraction – alcohol precipitation procedure:

Since RNA does not bind to the silica matrix, plasmid DNA can be separated from RNA during mini-prep workup.     This substitution saves time and reduces the use of harmful chemicals in the laboratory.  Using this method may reduce problems, such as incomplete RNA digestion, which make gels more difficult to interpret (see sample gel).
 
• Removal of restriction enzymes and cleaning of most reaction mixtures:

The silica matrix used in GENECLEAN^® does not  bind proteins or other organic compounds that may be left in solution, so GENECLEAN^® can serve as a quick method to clean DNA sample solutions without alcohol precipitation.
 
• Desalting reaction mixtures to change buffers:

GENECLEAN^® can be used to clean DNA samples that require different salt concentrations for buffer solutions.  The final cleaned DNA product is isolated in TE or water at very low salt concentrations.

The GENECLEAN^® kit provides the materials necessary to isolate DNA based on its binding properties to silica (glass) at different salt concentrations.  At high salt concentrations, sodium cations break hydrogen bonds between the hydrogens in water and the negatively charged oxygen ions in silica.  Sodium serves as a cation bridge and attracts the negatively charged oxygen in the phosphate backbone of the DNA molecule² (Figure 1).

Once the salt has been removed by rinsing the DNA-silica pellet several times, the DNA detaches from the silica and can be removed in solution.  The DNA is isolated in TE or water and does not need to be purified further.²

Volgelstein and Gillespie³ found that dissolving DNA cut from agarose gels in NaI (salt) can serve as a rapid, convenient method that yields high purity DNA solutions.  They suggest that “this method is particularly suitable for recovering DNA from agarose for further analysis or purification.”  They also found that glass powder gave the highest amount of bound DNA per weight of glass, compared to glass fiber and glass surfaces.

The silica included in the GENECLEAN^® kit, called GLASSMILK^®, consists of very small, irregularly shaped silica particles.  Bio101, Inc.⁴ claims that this matrix is advantageous over spherical or fibrous silica matrices.  GLASSMILK^® has a greater surface area, and thus can bind more DNA per unit volume than other regularly shaped silica matrices.  This matrix allows for more concentrated solutions of DNA after cleaning, because a smaller elution volume is needed to remove the salt from the DNA-silica pellet.

1.  Add 3X volume of NaI to the DNA solution and incubate at 45-55 C for five minutes if agarose is present.
2.  Add GLASSMILK^® according to kit guidelines and vortex for 1 minute.  Allow the solution to incubate at room temperature for 5 minutes.
3.  Pellet the silica matrix with the bound DNA according to kit guidelines.
4.  Wash the pellet 3 times with NEW wash.  Resuspend the pellet in NEW wash, spin for 5 seconds and discard the supernatant.
5.  Elute DNA from GLASSMILK^® by resuspending the pellet with water, TE or Elution Solution.  Centrifuge for 30 seconds and save the supernatant.  Elution may be repeated.

¹BIO 101:Protocols:DNA Kits:GENECLEAN.  1998 Jan 18.    <http://www.bio101.com/protocols/dna-kits/p-geneclean.html>  Accessed 1999 Feb 10.

²Bio 101, Inc.:Newsletters:Function:Issue 2:Page 3.  1998 Oct 28.    <http://www.bio101.com/newsletter/august98/3.html>  Accessed 1999 Feb 12.

³Volgelstein, B. and Gillespie, D.  Preparative and analytical purification of DNA from agarose.  Proc. Nat. Acad. Sci.  1979 Feb; 76(2):615-619.

⁴Bio 101, Inc.:Newsletters:Function:Issue 2:Page 4.  1998 Oct 7.   <http://www.bio101.com/newsletter/august98/4.html>  Accessed 1999 Feb 12.

© Copyright 2000 Department of Biology, Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: lafreeman@davidson.edu