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© 1999 PE Biosystems
User Bulletin: ABI 380B
Nucleic Acid Synthesizers
Subject: Using Wide-Pore, 1-µmol
DNA Synthesis Columns on the
Model 380B
User Bulletin - Number 72
Model 380B
Wide-Pore, 1 µmol Columns
Using Wide-Pore, 1-µmol DNA
Synthesis Columns on the Model
380B
Wide-Pore, 1-µmol DNA synthesis columns are useful for the synthesis of large
quantities of oligonucleotides greater than 50 bases. For many research,
diagnostic and clinical laboratories, GeneAmp PCR experiments have become a
routine tool for analysis, requiring larger amounts of primers to be made in one
synthesis. Some PCR experiments are designed for longer primers, with
restriction sites or regulatory regions built into them; others require a longer
primer (greater than 50 bases) with some degeneracy. For the latter, as
degeneracy increases, the amount of desired sequence in the solution decreases.
To ensure an adequate concentration of the desired sequence, an excess of the
degenerate primer is used.
Some longer primers and probes are used repeatedly in various experiments. If
these are not commercially available, using Wide-Pore, 1-µmol columns is a
convenient, cost-effective method of obtaining 150-300 optical density units of
oligonucleotide. These columns are similar to existing ABI synthesis columns in
both the SNAP design of the housing and the internal controlled-pore glass (CPG)
support. However, to accommodate the greater amount of 1000 Å CPG in the
column, the internal volume is slightly larger.
Optimized Wide-Pore, 1-µmol DNA Synthesis
Cycle
The yield and purity of oligonucleotides synthesized on the Model 380 B with
Wide-Pore, 1-µmol columns can be improved with only minor modifications to the
synthesis cycle. Because the internal volume of the column is larger, the reverse
flushes are extended, so flushing with argon is more complete. Reverse-flush
cycle times (ceaf3, Ver. 2.01) should be doubled from five seconds to ten (steps
3, 40, 45, 51, 58, 63 and 87). Cycle time will increase by approximately 50
seconds.
To Create a Special User Cycle:
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User Bulletin: ABI 380B Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/38x/ub/html/UB72.html
1. From the Main menu, choose Cycle and Procedure Editor.
2. Choose Synthesis Cycles/Edit Old.
3. Choose Cycle ceaf3 (Ver. 2.01) or the 1-µmol cycle commonly used.
4. Change the times by positioning the active field with the roll up/down,
left/right selections.
5. Name the cycle and then store it.
6. Print and verify the new cycle.
Product Information
Part numbers for the Wide-Pore, 1-µmol DNA Synthesis Columns are listed
below. All other synthesis reagents are the same as those used with existing ABI
DNA synthesis columns. The phosphoramidites should be diluted to 0.1 M for use
with these columns.
Part Number Description
401438 dAbz Wide-Pore, 1-µmol Column
401439 dGibu Wide-Pore, 1-µmol Column
401440 dCbz Wide-Pore, 1-µmol Column
401441 T Wide-Pore, 1-µmol Column
Information subject to change without notice.
Last updated May 1996
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2 of 2 11/23/99 8:30 AM
User Bulletin: ABI 380B Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/38x/ub/html/UB72.html
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