CLASSIC PROTOCOL

PUBLISHED IN ASSOCIATION WITH

COLD SPRING HARBOR LABORATORY PRESS

Gel-filtration chromatography
© 2006 Nature Publishing Group http://www.nature.com/naturemethods

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.

PROCEDURE

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

2| Wash the swollen resin several times with distilled sterile water to remove soluble dextran.
Soluble dextran can create problems by precipitating during ethanol precipitation.

3| Equilibrate the resin in Tris-EDTA buffer (TE; pH 7.6), autoclave at 10 p.s.i. (0.70 kg/cm2) for
15 min and store at 15–25 °C.

4| Prepare Sephadex or Bio-Gel columns in disposable 5-ml borosilicate glass pipets or Pasteur pipets Column
plugged with a small amount of sterile glass wool. Use a long, narrow pipet (for example, a disposable chromatography
1-ml plastic pipet) to push the wool to the bottom of the glass or Pasteur pipet.

5| Use a Pasteur pipet to fill the column with a slurry of the Sephadex or Bio-Gel, taking care to avoid
producing bubbles. There is no need to close the bottom of the column. Keep adding gel until it packs
to a level 1 cm below the top of the column. Wash the gel with several volumes of Tris-EDTA–NaCl
buffer (1× TEN; pH 8.0).

6| Apply the DNA sample (in a volume of 200 µl or less) to the top of the gel. Wash out the sample
tube with ~100 µl of 1× TEN buffer and load the wash buffer on the column as soon as the DNA
sample has entered the gel. When the washing has entered the gel, immediately fill the column with
1× TEN buffer.
▲ CAUTION Columns used to separate radiolabeled DNA from radioactive precursors should be run
behind Lucite screens to protect against exposure to radioactivity.

© Cold Spring Harbor Laboratory Press NATURE METHODS | VOL.3 NO.5 | MAY 2006 | 411
 CLASSIC PROTOCOL

7| Immediately start to collect fractions (~200 µl) in microcentrifuge tubes.

If the DNA is labeled with 32P, measure the radioactivity in each of the tubes by using either a
handheld minimonitor or by Cerenkov counting in a liquid scintillation counter. Add more 1× TEN
buffer to the top of the gel as required from time to time. The DNA will be excluded from the gel
and will be found in the void volume (usually 30% of the total column volume). The leading peak of
radioactivity therefore consists of radionucleotides incorporated into DNA and the trailing peak consists
of unincorporated [32P]dNTPs.
© 2006 Nature Publishing Group http://www.nature.com/naturemethods

8| Pool the (radioactive) fractions in the leading peak and store them at –20 °C.
SOURCE
This protocol was adapted from “Gel-filtration chromatography,” in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell,
D.W.) A8.29–A8.30 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001; http://www.cshlpress.com/link/
molclon3.htm).

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

412 | VOL.3 NO.5 | MAY 2006 | NATURE METHODS

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.