The RNA oligonucleotides (5′-UCAGΨCAGU-3′, 5′-ACUGACUGA-3′, 5′-UCACΨGAGU-3′, 5′-ACUCAGUGA-3′) were synthesized on an Applied Biosystems DNA/RNA synthesizer, deprotected and purified according to previously published procedures31 (link),53 (link),54 (link). For NMR study, each of the obtained duplexes was dissolved in a volume of 200 μl of 90% H2O and 10% D2O solution and placed into a 3 mm NMR sample tube (Supplementary Methods, Section 1 ).
Dna rna synthesizer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DNA/RNA synthesizer is a laboratory instrument designed to automate the synthesis of synthetic DNA and RNA molecules. It utilizes a series of chemical reactions to assemble nucleotides into desired oligonucleotide sequences.
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5 protocols using dna rna synthesizer
1
Synthesis and Purification of RNA Oligonucleotides
2
Synthesis and Characterization of Oligoribonucleotides
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All oligoribonucleotides: (AGG)2A, (AGG)4A, G(CGG)2C, G(CGG)4C, p(UGG)2U and p(UGG)4U were chemically synthesized on the Applied Biosystems DNA/RNA synthesizer using β-cyanoethyl phosphoramidite chemistry on solid support (36–38 ). The samples preparation procedure can be found in the Supplementary Data.
Our earlier experience with molecules composed of AGG, CGG and UGG repeats have shown that occasionally their spectroscopic features can be improved adding 5′-phosphate group. The role of 5′-phosphate has been discussed several times in the context of the G-quadruplex structures (39 (link),40 (link)). In this study, this type of modification was used only for UGG repeats, because it improved the quality of the 1H NMR spectra.
Our earlier experience with molecules composed of AGG, CGG and UGG repeats have shown that occasionally their spectroscopic features can be improved adding 5′-phosphate group. The role of 5′-phosphate has been discussed several times in the context of the G-quadruplex structures (39 (link),40 (link)). In this study, this type of modification was used only for UGG repeats, because it improved the quality of the 1H NMR spectra.
3
Crystallization of DNA Hexamer
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A DNA hexamer with the d(CGCGCG) sequence was synthesized on an Applied Biosystems DNA/RNA synthesizer using phosphoramidite chemistry. The methods of deprotection and purification of the oligodeoxynucleotides have been described previously [16 ]. A 1.5 mM water solution of the DNA oligonucleotide was annealed at 338 K for 12 min to form a DNA duplex. Single crystals of the DNA were grown at 292 K by the hanging-drop vapor diffusion method by mixing 2 μl of nucleic acid solution and 2 μl of precipitating solution containing 10 % (v/v) (±)2-methyl-2,4-pentanediol (MPD), 40 mM sodium cacodylate, pH 6.0, 80 mM KCl, 12 mM NaCl and 12 mM sperminium tetrachloride. The drops were equilibrated against 0.5 ml of 35 % (v/v) MPD. The crystals appeared within 1 week and grew to the dimensions of 0.2 × 0.1 × 0.1 mm. The best crystals were used for metal ion soaking. For metal soaking, the crystals were placed for several days in 2 μl of the reservoir solution mixed with 2 μl of 5 mM [Cr(H2O)6]Cl3.
4
Crystallization of RNA Oligomers
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The oligomer was synthesized by the solid phase method in an Applied Biosystems DNA/RNA synthesizer using TOM protected phosphoramidites. The synthesis was carried out in DMT-ON mode resulting in the oligomer carrying a trityl group at the 5′ end. The RNA was purified according to the protocol suitable for Glen-Pak cartridges (Glen Research) and then desalted and lyophilized under vacuum using a Speed-Vac. The purity of the oligomer was monitored using analytical HPLC separation (Lang and Micura 2008 (link)). Usually, the purity was at least 90%, which is suitable for crystallization. Before crystallization the RNA was dissolved in 10 mM sodium cacodylate pH 7.0 to a final concentration of 1 mM. The oligomer was denatured for 5 min at 95°C and cooled to ambient temperature within 2 h. Crystals were grown by the sitting drop method at 19°C. Most of the crystals grew under similar conditions: 0.08 M NaCl, 0.04 M sodium cacodylate pH 6.0, 45% MPD and 12 mM spermine. Depending on the metal ions the solution was supplemented with 20 mM BaCl2 or CdCl2 or SrCl2 or CaCl2 or CsCl. Only the unliganded structure was crystallized in 10 mM magnesium acetate, 50 mM MES pH 5.6 and 2.5 M ammonium sulfate. The RNA was mixed with the crystallization solution at a ratio of 3:1.
5
Synthesis of Modified Oligonucleotides
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The oligonucleotides were synthesized on a 1 μmol scale using a DNA automated synthesizer (Applied Biosystems, Waltham, MA, USA, 392 DNA/RNA Synthesizer) with conventional phosphoramidite chemistry. The reagents were purchased from Glen Research (Sterling, VA, USA) and Sigma-Aldrich, and the synthesis was conducted using ultra-mild phosphoramidites (phenoxyacetyl-dA (Pac-dA), Tac-dG, Ac-dC, T), with 0.25 M 5-benzylthio-1H-tetrazole (BTT) in CH3CN as an activator, 3% dichloroacetic acid (DCA) in CH2Cl2 as a deblocking solution, and 5% Tac2O and 16% N-methylimidazole in THF as a capping reagent. The ODNs incorporating the original NPu and OPu were synthesized as previously reported [31 (link)]. Fully natural ODNs were purchased from Japan Bio Service Co., Ltd. (Osaka Japan).
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