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Automated purification and quantification of oligonucleotides
K M Ivanetich1, R C Reid, R Ellison
1Biomolecular Resource Center, University of California, San Francisco 94143, USA. kathyi@cgl.ucsf.edu
Biotechniques
|October 19, 1999
Summary
Automated methods for trityl-on purification and quantification of synthetic oligonucleotides were developed. These methods enable efficient purification and accurate quantification, yielding successful primers for automated DNA sequencing.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Automated synthesis of oligonucleotides is crucial for molecular biology applications.
- Efficient purification and quantification are essential for reliable downstream use.
Purpose of the Study:
- To develop and validate automated methods for the trityl-on purification of synthetic oligonucleotides.
- To establish an automated system for the quantification of purified oligonucleotides.
Main Methods:
- Solid-phase extraction using Amberchrom CG-50 resin on an XYZ-axis robotic system for purification.
- Online UV-visible spectrophotometry (OD260nm) with a sipper for quantification.
Main Results:
- The automated purification method requires minimal user set-up time (5 min) and robot time (20 min per sample).
- An average yield of 28% was achieved for 15-25-mer oligonucleotides synthesized at the 40 nmol scale.
- Purified oligonucleotides were successfully employed as primers for automated DNA sequencing.
Conclusions:
- The developed automated methods provide an efficient and reliable approach for oligonucleotide purification and quantification.
- This automated workflow is suitable for producing high-quality oligonucleotides for applications such as automated DNA sequencing.