Waters Gen-Pak FAX Columns - Manual (Page 4)

[ Care and Use ManUal ]
Gen-Pak FAX Columns 4
The inclusion of EDTA in the buffers is recommended to protect the
DNA from nucleases during and after the chromatography1. Filter and
vacuum degas the buffers before use.
Useful chromatography can be obtained with sodium phosphate
buffer, but the phosphate coprecipitates with the DNA in the common
ethanol-precipitation procedures.
c. Equilibration and flow
Rigorous control of column equilibration is essential for predictable
and reproducible chromatography. Before injection, equlibrate the
column with 10 column volumes of buffer at initial chromatographic
conditions. The column can be used at flow rates from below 0.5 to
1 mL/min, but 0.75 mL/min is recommended for general use.
d. Temperature Effects
The conformation and intermolecular hydrogen bonding of DNA
fragments, and therefore the Gen-Pak FAX separation of those
fragments, are affected by temperature. At higher temperatures, the
structure of DNA is more open or relaxed so more ionic groups can
interact with the column. In general, higher ionic strength is required
as temperature is elevated.
Since conformation is dependent on base sequence, variations in
temperature over the range of 30 to 60 °C can be used to optimize a
particular separation. Where elevated temperatures are not required,
the column should still be operated at a controlled temperature,
typically 5 °C above ambient, to ensure reproducibility.
e. Detection
The separation of subnanogram quantities of DNA can be monitored
at 260 nm, provided that the buffers are pure and free of UV
absorbing contaminants. Assess blank gradients on a properly
equilibrated column prior to performing actual sample
chromatography.
f. Sample Size
Depending upon the size differences among the fragments to be
isolated, as much as 50 to 100 μg of DNA can be applied to the
column. Injection volume is essentially unlimited as long as the ionic
strength of the sample is at least 0.1 M less than that required to
elute the fragment of interest. For injection volumes above 100 μL,
the sample should be at pH 8.
Excellent recoveries, in most cases more than 95 percent, of
biologically active material have been obtained purifying samples
containing fragments of 5000 base pairs and less.
g. Sample Treatments
DNA fragments generated by restriction enzyme digestion or
from the polymerase chain reaction may be extracted with phenol/
chloroform prior to chromatorgraphy. However, the reaction may be
injected directly since most proteins are completely unretained under
conditions used for the separation (i.e., 0.30 M NaCl).
In either case perform these steps before injection:
1. Remove particulate material from samples by centrifugation or
filtration through a 0.45 µm filter.
2. Heat treated samples at 37 °C for 10 min prior to injection to
disrupt any reannealed termini of the DNA fragments.
h. Gradients
Typical DNA fragments elute from the Gen-Pak FAX column between
0.4 and 0.75 M NaCl at 30°C. Equilibrate the column to 0.1 M below
the ionic strength required to elute the smallest fragment of interest.
Useful gradients are usually 5 to 10 mM/min at 0.75 mL/min.
i. Column Washing
Upon completion of each separation, wash the column with a small
volume of 20 - 40% acetic acid to maintain its chromatographic
characteristics as well as to eliminate sample carry-over from injec-
tion to injection.
Procedure
After the last fragment elutes, perform the following procedure:
1. Flush the column with 5 mL of buffer B.
2. Pump or inject 3 to 5 mL of 20 - 40% acetic acid onto the
column. This may be performed with the column at 60 °C.
3. Flush the column with about 5 mL of buffer B.
4. Reequilibrate the column for the next sample injection.
Note: Washing the column with sodium hydroxide does not perform as
well as the above acetic acid procedure and is not recommended.