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Molecular Biology GradeAG® 50W-X8
Cation Exchange Resin
Instruction ManualLIT304B

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Table of Contents
Section 1

Introduction ..........................................1

Section 2

Description ............................................2

Section 3

Instructions for Use ..............................3

Section 4

Sample Protocol ....................................4

4.1
4.2

Material Required ...........................................4
Protocol ...........................................................5

Section 5

Reference ...............................................7

Section 6

Production Information .......................7

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Section 1
Introduction
Ethidium bromide is used to visualize DNA andRNA preparations because it intercalates between thebases and fluoresces when irradiated with UV light at
300 nm. Propidium iodide, which also intercalates
between bases, is used to increase the separation ofsuperhelical and non-supercoiled DNA in cesium
chloride gradients. Molecular Biology Grade AG 50WX8 cation exchange resin is useful for removing thesedyes, to provide a purified plasmid preparation. The
resin is certified to be free of endo-and exo-nuclease,and ligase inhibitors. The resin has a high selectivity forthe hydrophobic, cationic ethidium and propidium ionsand repels the strongly anionic nucleic acid. Thefluorescent labeled DNA is passed over a column of theMolecular Biology Grade AG 50W-X8 cation exchange
resin, which removes the fluorescent dye from the DNAor RNA. The pure plasmid preparation is then collectedin the void volume.
1

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Section 2Description

RAG 50W-X8 cation exchange resin is the backbone
resin of the Molecular Biology Grade AG 50W-X8resin. AG 50W-X8 resin is a strong cation exchanger
with sulfonic acid functional groups attached to a styrene
divinylbenzene copolymer lattice. The MolecularBiology Grade resin is provided in the sodium form. It
has been specially equilibrated in Tris buffer at pH 8.0.
The resin is capable of exchanging cations of salts, andof ampholytes on the acidic side of their pI. This resin
can be used to extract basic compounds from a solution
with a pH at least 1 unit lower than the pK a of theanalyte. The analytes are then eluted with a neutral orbasic pH solution.

2SO3H2NA

NH2BrN+CH2CH3RSO3H2N

B

NH2CH3ZIN+
CH2CH2CH2N1—CH2CH3CH2CH3Fig. 1. Removal of (A) ethidium bromide and (B) propidium
iodide by AG 50W-X8 cation exchange resin. R is the resin matrix;
SO3- is the functional group covalently attached to the resin.

Section 3
Instructions for Use
For removing ethidium and propidium salts fromplasmids, the resin is slurried in a pH 8.0 Tris buffer andpoured into a column. The sample pH is adjusted to pH
8.0. Thus the DNA will be anionic, and ethidium andpropidium will be cationic. When the sample is added tothe column, the ethidium and propidium are exchanged
for sodium ions on the resin. The pure plasmid
preparations pass through the column immediately (inthe void volume) and the fluorescent dyes are bound to3

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Page 4the resin. Figure 1 shows the removal of (A) ethidium
bromide and (B) propidium iodide by Molecular Biology
Grade AG 50W-X8 cation exchange resin. R is the resin
matrix; SO3- is the functional group covalently attachedto the resin.

Section 4
Sample Protocol

500 ml beaker
Test tube rack for Poly-Prep® chromatography
columns
Test tubes or other containers to collect DNA

4.2 Protocol

The following procedure was developed byRodriquez and Tait to remove propidium iodide from
intact plasmids, and is used for DNA concentrated on
cesium chloride gradients.1

4.1 Material required
Concentrated HCl
A-50 buffer (4 liters):
Trizma baseNaCl
0.25 M EDTA
1 M NaN3
Distilled water

Adjust pH to 8.0 with 8-10 ml concentrated HCl.
Add distilled water to 4.0 liters.

24 grams (50 mM)
117 grams (500 mM)
16 ml (1 mM)
4 ml
3.5 liters
41. Use about 1.5 ml (approximately 1.1 grams) of
Molecular Biology Grade AG 50W-X8 resin in acolumn for each gradient tube. The resin may also beautoclaved in the Poly-Prep chromatography
columns, if a sterile preparation is desired.
2. Slurry the resin for each column to be poured in 2.3
volumes of A-50 buffer. Pour all at once into a PolyPrep column. Do not snap the tip off the column until
ready to proceed with Step 3. At this time, the resinand column may be autoclaved.
3. Snap the tip off the column and wash the column
with 20 ml of A-50 buffer.
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4. Dilute DNA with an equal volume of A-50 buffer
and apply the DNA sample to the column. Dilutionof the sample is necessary to slow the flow rate andallow complete removal of the propidium orethidium. Use a stopcock to slow the flow rate ifnecessary.
5. Add 1 bed column (1.5 ml) of A-50 buffer to wash
out any remaining DNA.
6. Check DNA sample with UV to insure that the dye
has been removed. If not, remove the top layer of theresin containing the dye, discard, and run the DNA
sample over the column again.

Section 5Reference

Warning: Ethidium and propidium are carcinogenic.
The used columns should be disposed of as hazardous
waste.

143-1441
143-1451

1.

Rodriquez, R. L. and Tait, R. C., Recombinant DNA
Techniques: An Introduction, Addison-Wesley PublishingCo., Reading, Massachusetts, 153-158 (1983).

Section 6
Product Information
Catalog
Number

Ionic
Form

Wet
DensityPkg. Capacity Diameter (Nominal)
Size (meq/ml) (microns) gm/ml

Molecular Biology Grade AG 50W-X8 Resin
100-200
200-400

Sodium 100 gSodium 100 g

1.7
1.7Columns
731-1550
731-7005

6MeshSize

Poly-Prep® Columns, empty, 50
Poly-Prep Column Rack, 1

7106-250
63-1500.80
0.80

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Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
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