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INCORPORATION OF AZOBENZENES INTO DNA

Method of Incorporation of Azobenzene into DNA: 概要

CONFIRMATION THAT AZOBENZENE PORTFOLIO IS INCLUDED IN DNA.

Method of Incorporation of Azobenzene into DNA: ようこそ！

DNA SYNTHESIS

  1.  Dissolve the dried compound 3  (250 mg, 0.307 mmol) in dry AN (3.1 ml) in a vial (the concentration of compound 3  is approximately 0.1 M). With this amount of compound 3 , approximately 3 X  residues can be incorporated through the DNAsynthesizer using a synthesis program of 0.2 m mol scale.

  2.  Equip the DNA synthesizer with the vial containing the solution of compound 3  as well as with vials of solutions ofconventional dA, dG, dC and dT phosphoramidite monomers.

  3. Set the 3’-Dabcyl-CPG support (for Dabcyl-DNA) or dT-CPG, C6-NH2-CPG on the DNA synthesizer and start the synthesis at a scale of 0.2 mmol.

  4. Stop the DNA synthesis; do not remove the DMT group at the 5'-terminal. Full-length DNA with the X insert will thus have been synthesized on the CPG support. 

Method of Incorporation of Azobenzene into DNA: 概要

DEPROTECT AND PURIFICATION

  1. Transfer the CPG support to a vial and add 500µl AMA.

  2. It was heated at 65˚C for 10 mins for the deprotection of phosphate group and nucleic acid base and cleavage from the CPG support at the same time.

  3. Added Nacl 500µl (100mg/ml)

  4. Flushed the glen-Pak cartridge with 1ml 2 M TEAA.

  5. Loaded the DNA solution onto the cartridge. Collect the eluted fraction

  6. Flushed the glen-Pak cartridge with1ml sample

  7. Flushed the glen-Pak cartridge with1ml Salt wash solution (100mg/mL Nacl in 5% AN)

  8. Detritylated the support-bound DNA by flushing the cartridge with 1ml 2% TFA solution twice.

  9. Flushed the glen-Pak cartridge with 1ml mQ water.

  10. Eluted the purified, detritylated DNA by flushing the cartridge with 50% AN.

  11. Removed the solvent using a vacuum dryer.

  12. After simple purification using NAP column and evaporation of solvent.

Method of Incorporation of Azobenzene into DNA: 概要

IDENTIFICATION AND PURIFICATION

  1. Characterize the sample by MALDI-TOFMS of the DNA solution using 1:1water/AN solution of 3-hydroxypicolinic acid and 0.5 M diammonium hydrogen citrate solution as a matrix.

  2. Purify  the stock solution by HPLC.

  3. Characterize the sample by MALDI-TOFMS of the DNA solution using 1:1water/AN solution of 3-hydroxypicolinic acid and 0.5 M diammonium hydrogen citrate solution as a matrix.

Method of Incorporation of Azobenzene into DNA: 概要

STAPLE DNA INCORPORATED INTO TIRES

Method of Incorporation of Azobenzene into DNA: ようこそ！

DNA SYNTHESIS

  1. Transfer the CPG support to a vial and add 500µl AMA. 

  2. Dissolve the dried compound 3  (250 mg, 0.307 mmol) in dry AN (3.1 ml) in a vial (the concentration of compound 3  is approximately 0.1 M). With this amount of compound 3 , approximately 4 X  residues can be incorporated through the DNAsynthesizer using a synthesis program of 0.2 m mol scale.

  3.  Equip the DNA synthesizer with the vial containing the solution of compound 3  as well as with vials of solutions ofconventional dA, dG, dC and dT phosphoramidite monomers.

  4. Set the 3’-Dabcyl-CPG support (for Dabcyl-DNA) or dT-CPG, dC-CPG,  dT-CPG, dA-CPG on the DNA synthesizer and start the synthesis at a scale of 0.2 mmol.

  5. Stop the DNA synthesis; do not remove the DMT group at the 5'-terminal. Full-length DNA with the X insert will thus have been synthesized on the CPG support.

Method of Incorporation of Azobenzene into DNA: 概要

DEPROTECT AND PURIFICATION

  1. It was heated at 65℃for 10 mins for the deprotection of phosphate group and nucleic acid base and cleavage from the CPG support at the same time.

  2. Added Nacl 500µl (100mg/ml)

  3. Flushed the glen-Pak cartridge with 1ml 2 M TEAA.

  4. Loaded the DNA solution onto the cartridge. Collect the eluted fraction

  5. Flushed the glen-Pak cartridge with1ml sample

  6. Flushed the glen-Pak cartridge with1ml Salt wash solution (100mg/mL Nacl in 5% AN)

  7. Detritylated the support-bound DNA by flushing the cartridge with 1ml 2% TFA solution twice.

  8. Flushed the glen-Pak cartridge with 1ml mQ water.

  9. Eluted the purified, detritylated DNA by flushing the cartridge with 50% AN.

  10. Removed the solvent using a vacuum dryer.

  11. After simple purification using NAP column and evaporation of solvent.

Method of Incorporation of Azobenzene into DNA: 概要

IDENTIFICATION AND PURIFICATION

  1. Characterize the sample by MALDI-TOFMS of the DNA solution using 1:1water/AN solution of 3-hydroxypicolinic acid and 0.5 M diammonium hydrogen citrate solution as a matrix.

  2. Purify  the stock solution by HPLC.

  3. Characterize the sample by MALDI-TOFMS of the DNA solution using 1:1water/AN solution of 3-hydroxypicolinic acid and 0.5 M diammonium hydrogen citrate solution as a matrix.

Method of Incorporation of Azobenzene into DNA: 概要
Method of Incorporation of Azobenzene into DNA: Service
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