References
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- A Cruachem semi-manual oligonucleotide synthesizer with an Apple IIe computer for timed step-by-step instructions was instructions was used for the preparation of the oligomers with the phosphotriester method. For the instructions a published protocol was followed7 with the exception of doubling the coupling time with II
- FPLC (Fast Protein Liquid Chromatogaraphy) purification and analyses were carried out on a Pharmacia system consisting of a GP 250 gradient programmer, 2 P-500 pumps, a 5 MP mixer and a V-7 10 MPa injector with a MonoQ HR 5/5 anion exchange column
- The yield of unlabeled d(T)15 was 12 to 15 OD after FPLC purification. Therefore, the majority of the observed loss was not due to sample handling but caused by a reduced coupling efficiency in the presence of the nitroxide
- EPR spectra were measured on a Bruker ESP 300 or a Varian E-104 spectrometer.
- Borer , P. N. 1975 . “Handbook of Biochemistry and Molecular Biology, Nucleic Acid I” , Edited by: Fasman , G. D. 589 CRC Press .
- Atkinson , T. and Smith , M. 1984 . “Oligonucleotide Sunthesis: A Practical Approach” , Edited by: Gait , M. J. 35 IRL Press .
- Caruthers , M. H. , Barone , A. D. , Beaucage , S. L. , Dodds , D. R. , Fisher , E. F. , McBride , L. J. , Matteucci , M. , Stabinsky , Z. and Tang , J.-Y. 1987 . Methods Enzymol. , 154 : 287
- Oligonucleotide synthesis with the phosphoramidites was performed on a fully automated Pharmacia Gene Assembler DNA Synthesizer using the Pharmacia Protocol Expect for about doubling the coupling time with III.
- We determined that the nitroxide undergoes a 10 to 20% destruction during each oxidation step with 0.1M I2 in H2O/2,6-lutidine/THF. 13 While the phosphoramidite protocol for the Applied Biosystems 380A or 380B instrument requires 0.1M I2, the Pharmacia protocol calls for only 0.01M I2. EPR experiments with this tenfold lower I2 concentration showed a considerably smaller nitroxide destruction