Automated purification and quantification of oligonucleotides.
We have developed automated methods for the trityl-on purification and quantification of synthetic oligonucleotides. Oligonucleotide purification is by solid-phase extraction cartridges using Amberchrom CG-50 resin on an XYZ-axis robotic system. Quantification is by OD260nm using an online UV-visible spectrophotometer with sipper. The purification of 20 oligonucleotides requires 5 min of user set-up time, plus 20 min per sample of robot time. For a 15-25-mer at the 40 nmol scale of synthesis, the method gives a yield of 2.8 ODs from a load of 10.1 OD, i.e., a 28% average yield. Oligonucleotides purified by this method have proven to be successful for primers for automated DNA sequencing.
Ivanetich
K M
KM
Biomolecular Resource Center, University of California, San Francisco 94143, USA. kathyi@cgl.ucsf.edu
Reid
R C
RC
Ellison
R
R
Perry
K
K
Taylor
R
R
Reschenberg
M
M
Mainieri
A
A
Zhu
D
D
Argo
J
J
Cass
D
D
Strickland
C
C
D001322:Autoanalysis; D001483:Base Sequence; D002845:Chromatography / Q000191:economics / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003365:Costs and Cost Analysis; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis* / Q000138:chemical synthesis / Q000302:isolation & purification; D010957:Plasmids; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet; D013698:Templates, Genetic; D013997:Time Factors
D009841:Oligonucleotides