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Gel-filtration chromatography

Nature Methods volume 3, pages 411–412 (2006)Cite this article

Exclusion chromatography can be used to separate molecules on the basis of size1,2. The technique described here, which uses gel filtration to separate high-molecular-weight DNA from smaller molecules, is used most often to separate unincorporated labeled dNTPs from DNA that has been radiolabeled. It is also used at several stages during the synthesis of double-stranded cDNA, during addition of linkers to blunt-ended DNA, to remove oligonucleotide primers from PCR and, in general, whenever it is necessary to change the composition of the buffer in which DNA is dissolved. The two most commonly used gel matrices are Sephadex and Bio-Gel, both of which are available in several porosities. Sephadex G-50 and Bio-Gel P-60 are ideal for purifying DNA larger than 80 nucleotides in length. Smaller molecules are retained in the pores of the gel, whereas the larger DNA is excluded and passes directly through the column. Bio-Gel P-2 can be used to separate oligonucleotides from phosphate ions or dNTPs. Bio-Gel is supplied in the form of a gel and only needs to be equilibrated in running buffer before use. Sephadex is supplied as a powder that must be hydrated before use.

1 | Slowly add Sephadex of desired grade to distilled sterile water in a 500-ml beaker or bottle (10 g of Sephadex G-50 granules yields 160 ml of slurry; follow the manufacturer's instructions).

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References

  1. Barth, H.G., Boyes, B.E. & Jackson, C. Size exclusion chromatography. Anal. Chem. 66, 595R–620R (1994).

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  2. Northrop, D.N., Scott, R.P.W. & Martire, D.E. Preparation and evaluation of a bimodal size-exclusion chromatography column containing a mixture of two silicas of different pore diameter. Anal. Chem. 63, 1350–1354 (1991).

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Gel-filtration chromatography. Nat Methods 3, 411–412 (2006). https://doi.org/10.1038/nmeth0506-411

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