Oligonucleotides solution preparation

Oligonucleotide solutions are prepared in TE buffer pH 8 (0.1 M Tris-HCl buffer solution, ImM in EDTA). Aliquots are prepared and maintained at -20°C. Working solutions were conserved at 4°C. Hybridisation takes place in a 2 x SSC (saline sodium citrate, 30 mM sodium citrate buffer with 300 mM sodium chloride and pH 7.0) buffer containing 50% of formamide. 

Stock solutions (lmg/ml) of the oligonucleotides were prepared and kept frozen. The washing and measurement buffer was ABS. The dilution and hybridisation buffer was the 2 x SSC. 

Experimental details 241 DNA duplexes were formed from oligonucleotides and prepared on a DNA synthesizer by slow cooling of equal concentrations of complementary strands. Solutions of a duplex (8 jom), [Ru(phen)2(dppz)]2+ (8 iM) and 10 20 equivalents of a quencher (e.g. methyl viologen), were irradiated at 436 nm with an Hg Xe lamp (1000 W) equipped with a monochromator (Figure 3.28). After irradiation, the samples were treated with piperidine, dried and electrophoresed through polyacrylamide gel. The extent of DNA damage was evaluated by phosphorimaging. 

Fig. 1. Schemes for combination of protected oligonucleotide blocks prepared by phosphotriester solution synthesis (from 8). Symbols [BLlg base-labile-protecting groupil AL] = acid-labile-protecting group -I I -fully protected oligodeoxy-...

Each primer is a synthetic oligonucleotide of about 20 bases prepared so that then-sequences are complementary to the (previously determined) sequences that flank the tar get regions on opposite strands Thus one primer is annealed to one strand the other to the other strand The 3 hydroxyl end of each primer points toward the target region The stage is now set for DNA synthesis to proceed from the 3 end of each primer [Figure 28 14(c )] The solution contains a DNA polymerase and Mg " m addition to the... 

The solution structures of 1 1 complex between paromomycin and gentamicin Cla and the A-site oligonucleotide have been solved at high resolution using heteronuclear NMR techniques These approaches require the preparation of uniformly labeled N- and C-RNA oligonucleotides via in vitro transcription with labeled nucleoside triphosphates. The use of RNAs labeled with NMR-active nuclei allows for the application of sophisticated heteronuclear NMR methods. These methods facilitate the assigmnent of NMR resonances and the acquisition of structural restraints for detailed structure determinations. 

Prepare 10-20 pg of amine-containing oligonucleotide in 200 pi of water. Add to this solution 20 pi of 1 M sodium bicarbonate, pH 9. 

The majority of fluorescent probes are water-insoluble and must be dissolved in an organic solvent prior to addition to an aqueous reaction medium containing the DNA to be labeled. Suitable solvents are identified for each fluorophore, but mainly DMF or DMSO are used to prepare a stock solution. Some protocols utilize acetone when labeling DNA. However, avoid the use of DMSO for sulfonyl chloride compounds, as this group reacts with the solvent. For oligonucleotide labeling, the amount of solvent added to the reaction mixture should not exceed more than 20% (although at least one protocol calls for a 50% acetone addition—Nicolas etal., 1992). 

The 2 x SSC buffer solution contained 300 mM of NaCl and 30 mM C3H5Na307 (pH 7.4). The acetate buffer solution (ABS) was prepared at the final concentration of 0.25 M, with 10 mM of NaCl (pH 4.7). All oligonucleotides, probes and complementary strands were purchased from Sigma Genosys (UK). All measurements were performed using screen-printed electrodes (SPEs). SPEs were used as disposable. The three-electrode system used was formed by a graphite working electrode, a counter electrode and a silver-based reference electrode. 

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