Bio-Rad Bio-Gel A Agarose Gel - Manual (Page 6)

agents should be avoided because they react with the gel
and increase the content of charged groups on the matrix.
Bio-Gel A gels are generally not intended for use with
organic solvents, but will tolerate gradual exchange with
aqueous and dilute organic solutions up to 20%.
Section 4
Column Packing and Operation
Section 4.1 Column Packing
1. Bio-Gel A gel is supplied fully hydrated with sodium
azide as a preservative. The gel should be washed prior
to packing into a column. Pour the desired amount of
gel into a Buchner funnel equipped with a filter.
Apply a vacuum to remove excess water, then wash
the gel with 2–3 volumes of the buffer.
2. Slurry the prepared matrix in several bed volumes of
the running buffer and degas under vacuum for
5 minutes. Occasional swirling of the container will
help to fully degas the slurry. A stir bar should not be
used for this process.
3. Fill the column approximately 1/3 full with degassed
starting buffer and remove any air bubbles that might
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Section 3.2 Column Selection
A properly executed separation will usually employ a
bed with a length to diameter ratio between 5 and 10.
The bed volume employed is usually 4 to 10 times the
volume of the sample. The minimum anticipated dilution
factor for an excluded substance approaches 1.25.
Fractionation procedures generally require bed length to
diameter ratios of 25 to 100 or greater and bed volumes
25 to 100 times the sample volume. If beds in excess of
1 meter long are needed, two or more short columns may
be connected in series by flow adaptors.
Section 3.3 Eluant Selection
The eluant chosen should provide maximum stability
for labile sample solutes. To eliminate the effect of small
amounts of negatively charged groups on the gel, the
ionic strength should be at least 50 mM. Using highly
concentrated salt solutions greater than 500 mM may
cause the gel bed to shrink, as well as alter the exclusion
limit of the gel.
The recommended pH range is from pH 4.0 to pH 13.0.
Prolonged operation outside these limits may lead to
structural disruption of the gel beads. Strong oxidizing
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