Waters Gen-Pak FAX Columns - Manual (Page 3)

[ Care and Use ManUal ]
Gen-Pak FAX Columns 3
c. Operating Pressure
Do not exceed 4,000 psi operating pressure or about
1 mL/min at 25 °C.
d. pH range
Stay within a pH range of about 1.5 to 12 (do not use concentrated
acids or bases).
e. Flow
Make flow rate changes in a gradual manner (less than 1 mL/min) to
avoid column voiding. Never reverse flow in the column.
f. Cleaning
Clean the column between nucleic acid injections with 3 to 5 mL of
with 3 to 5 mL of 20 - 40% acetic acid.
g. Storage
When you store a column for less than 24 hours, you typically do
not need to follow special storage procedures. However, be sure
that the column never dries out; this can degrade chromatographic
performance.
For longer term storage, follow the procedure described below:
1. Flush the column with approximately 25 mL of Milli-Q ® water to
remove salts.
2. Flush the column with approximately 4 mL of a mixture of 10
percent methanol and 90% Milli-Q water.
3. Disconnect the column.
4. Screw end plugs firmly in place and return the column to its box.
5. Store the column at 4 °C.
Note: Before reusing the column, flush 4 with 25 mL of Milli-Q water
to remove any methanol prior to introducing buffers.
IV. GeneRAL MetHoD DeVeLoPMent GUIDeLInes
This section discusses the use of Waters Gen-Pak FAX columns for
purifying DNA restriction fragments, polymerase chain reaction (PCR)
products, plasmids, and synthetic oligonucleotides.
a. Separating Double Stranded DNA Fragments
DNA fragments are usually isolated using gel electrophoresis.
Although resolution is good, the technique has limited mass capacity,
often gives low yields of extracted fragments, and is time consuming.
The Gen-Pak FAX column is a useful alternative for the
rapid purification and analysis of such nucleic acid species.
Separations are often accomplished in about 30 minutes. Recoveries
of biologically active material directly from the column are usually
greater than 95%. Direct UV monitoring of the column effluent
provides subnanogram sensitivity without the need for indirect
visualization via ethdium bromide staining or autoradiography.
Depending upon sample complexity, as much as 50 to 100 μg
of DNA can be separated in a single run. Since the separation is
based primarily upon the overall charge of each fragment, smaller
fragments elute prior to larger ones using an ionic strength gradient
as shown in Figure 2.
Figure 2: Separation of 3.0 μg BstN I Digest of pBR322 DNA
b. Buffers
The preferred chromatographic buffer is 25 mM Tris/Cl. 1mM EDTA,
pH 8.0. The recommended buffers for DNA fragmet separations are:
• Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
• Buffer B: 25 mM Tris/Cl, mM EDTA, 1.0 M NaCl, pH 8.0
Column: Gen-Pak FAX ™ (4.6 x 100 mm)
Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
Buffer B: 25 mM Tris/Cl, 1 mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: 30 to 100% B in 30 min., linear
Flow: 0.75 mL/min
Temperature: 30˚C
0.30
0
18 35 Minutes
121 bp
382 bp
1060 bp 929 bp
1857 bp
Column: Gen-Pak FAX (4.6 x 100 mm)
Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
Buffer B: 25 mM Tris/Cl, 1 mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: 30 to 100% B in 30 min., linear
Flow: 0.75 mL/min
Temperature: 30˚C
Absorbance 260 nm
0.30
0
18 35 Minutes
121 bp
382 bp
1060 bp 929 bp
1857 bp