ONs 1–8, 10–13 and 15–23 were synthesized on a 394 ABI DNA/RNA synthesizer using controlled pore glass (CPG) supports and 2΄-O-t-butyldimethylsilyl-phosphoramidites (purchased from Glen Research). 0.25M 5-Ethylthio-1H-Tetrazole in Acetonitrile was used as the coupling reagent, 3% dichloroacetic acid in dichloromethane was used for de-tritylation, a 2,6-lutidine/acetic anhydride solution was used for capping and an I2/tetrahydrofuran/pyridine solution was used in the oxidation step. Coupling times of 10 min were used. ONs were deacetylated/debenzoylated and cleaved from the CPG support in the presence of 1:1 aq. methylamine (40%) and aq. ammonia (40%) with heat (60°C, 1.5 h). A mixture of N-methylpyrrolidinone/triethylamine/hydrogen fluoride (3:2:1) was used for deprotection of the tert-butyldimethylsilyl groups (60°C, 1 h) followed by purification via electrophoresis [20% denaturing polyacrylamide gel electrophoresis (PAGE)]. C18-Sep-Pak cartridges were obtained from Waters and used to desalt the purified oligomers using 5 mM NH4OAc as the elution buffer. ONs were redissolved in H2O and used as obtained for subsequent experiments. ONs 9 and 14 were purchased from IDT-DNA and, following quantification via UV-vis, used without further purification.
Abi 394 dna rna synthesizer
Manufactured by Glen Research
The ABI 394 DNA/RNA synthesizer is a laboratory instrument designed for the automated synthesis of DNA and RNA oligonucleotides. It utilizes phosphoramidite chemistry to assemble nucleic acid sequences in a controlled and efficient manner.
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7 protocols using abi 394 dna rna synthesizer
1
Synthesis and Purification of Oligonucleotides
2
Oligonucleotide Synthesis and Purification
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Oligonucleotides were synthesized on a 394 ABI DNA/RNA synthesizer using CPG supports and 2’-O-TBDMS phosphoramidites (purchased from Glen Research). 0.25M 5-Ethylthio-1H-tetrazole in acetonitrile was used as the coupling reagent; 3% trichloroacetic acid in dichloromethane was used for deblocking; a 2,6-lutidine/acetic anhydride solution was used for capping; and an iodine (0.02 M) in/THF/pyridine/water solution was used in the oxidation step (also purchased from Glen Research). Coupling times of 10 min were used. Oligonucleotides (ONs) were deacetylated / debenzoylated / deformylated and cleaved from the CPG support in the presence of 1:1 aq. methylamine (40%) and aq. ammonia (40%) with heat (60° C, 1.5 h). A mixture of N-methylpyrrolidinone/triethylamine/HF (3:2:1) was used for deprotection of the TBDMS groups (60° C, 1 h) followed by purification via electrophoresis (20% denaturing PAGE). C18-Sep-Pak cartridges were obtained from Waters and used to desalt the purified oligomers using 5 mM NH4OAc as the elution buffer. Oligonucleotides were dissolved in H2O and used as obtained for subsequent experiments. Unmodified ONs were purchased from IDT-DNA or ChemGenes and, following quantification via UV-vis, used without further purification.
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Oligonucleotide Synthesis and Purification
4
FLT3-ITD Antisense Oligonucleotide Design
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For FLT3‐ITD ASO, a series of 16‐nucleotide (nt) ASOs were designed to specifically target the repeated region of FLT3‐ITD. The ASOs have a 3‐10‐3 gapmer configuration (i.e. 3‐nt locked nucleic acid [LNA]‐modified ends flanking a central 10‐nt DNA segment) and are fully modified with phosphorothioate (PS) chemistry throughout the backbone. All FLT3‐ITD ASOs were synthesized in‐house with an ABI 394 DNA/RNA synthesizer on Glen UnySupport (Glen Research) using standard phosphoramidite chemistry. LNA phosphoramidites purchased from Sigma‐Aldrich, and phenylacetyl disulfide (ChemGenes Corporation) was used as the sulfurizing reagent. Oligonucleotide cleavage and deprotection were performed under concentrated aqueous ammonia at 55°C for 16 h. The ASOs were purified with Poly‐Pak II cartridges (Glen Research) following the manufacturer's protocol, desalted using Glen Pak 2.5 desalting column (Glen Research), and dried by lyophilization. The ASOs were characterized by JEOL SpiralTOF matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometer.
miR‐125b ASO and FAM‐conjugated ASO (FAM ASO) were synthesized by Shanghai Genepharma (Shanghai, China) as described in our previous paper.19
miR‐125b ASO and FAM‐conjugated ASO (FAM ASO) were synthesized by Shanghai Genepharma (Shanghai, China) as described in our previous paper.
5
Oligonucleotide Synthesis and Purification
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DNA oligonucleotides were chemically synthesized on an ABI 394 DNA/RNA synthesizer using reagents from Glen Research. The oligonucleotides were de-protected using ammonium hydroxide and purified with Poly-PakTM cartridges. DNA samples were successively dialyzed against water and 20 mM KCl solution. They were subsequently lyophilized and dissolved in a buffer containing 20 mM potassium phosphate (pH 7.0) and 20 mM KCl.
6
DNA Oligonucleotide Synthesis and Purification
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Unlabeled and site-specific labeled DNA oligonucleotides were chemically synthesized on an ABI 394 DNA/RNA synthesizer using products from Glen Research and Cambridge Isotope. DNA was purified by a reverse-phase Poly-Pak cartridge (Glen Research). Samples were then dialyzed successively against 25 mM KCl solution and against water. DNA oligonucleotides were dissolved in solution containing 70 mM KCl and 20 mM potassium phosphate (pH 7.0). DNA concentrations were determined by measuring the UV absorbance at 260 nm.
7
Oligonucleotide Synthesis and Purification
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Oligonucleotides were synthesized on ABI 394 DNA/RNA synthesizer using product from Glen Research and Cambridge Isotope Laboratories. Samples were then purified following Glen Research Protocol and dialyzed successively again water, 10 mM Tris pH 7.0, and water.
Next, samples were dried in lyophilizer, and then dissolved in a buffer containing 10 mM Tris (pH 7.0).
Next, samples were dried in lyophilizer, and then dissolved in a buffer containing 10 mM Tris (pH 7.0).
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