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IncorporationofPhosphoramiditeCompoundintoDNA…：的亚磷酰胺化合物掺入到DNA….doc
Phosphoramidite Compound
Incorporation of phosphoramidite compound into DNA
1. Dissolve the dried phosphoramidite compound (0.307 mmol) in dry AN (3.1 ml) in a vial (the concentration of phosphoramidite compound is approximately 0.1 M). With this amount of phosphoramidite compound, approximately 14 X residues can be incorporated through the DNA synthesizer using a synthesis program of 1.0 μmol scale.
2. Equip the DNA synthesizer with the vial containing the solution of phosphoramidite compound as well as with vials of solutions of conventional dA, dG, dC and dT phosphoramidite monomers.
3. Set the 3’-Dabcyl-CPG support (for Dabcyl-DNA) or dA-CPG (for non-template) on the DNA synthesizer and start the synthesis at a scale of 1.0 μmol.
CRITICAL STEP At the coupling step for the X residue, the reaction time should be 300 s or longer. If the phosphoramidite monomer works efficiently, the waste solvent at the detrityl step is orange and the CPG support also becomes orange because of the incorporated phosphoramidite compound.
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.
5. Transfer the CPG support to a vial and add 1 ml 28% aqueous ammonia.
PAUSE POINT Keep the vial at 50 ℃ with the thermostat overnight (approximately 12 h) to deprotect and cleave the DNA from the support.
6. Connect a syringe to the female luer of the Poly-Pak cartridge and have the male luer terminate in a waste vessel.
7. Flush the Poly-Pak cartridge with 4 ml AN followed by 4 ml 2 M TEAA.
8. Add 3 ml deionized water to the deprotected DNA in the ammonia solution.
9. Load the solution from Step 8 onto the cartridge. Collect the eluted fraction and reload it twice.
10. Flush the cartridge with 6 ml 1.3% aqueous ammonia solution.
11. Flush the cartridge with 4 ml deionized water.
12. Detritylate the support-bound DNA by flushing the cartridge with 4 ml 2% TFA solution.
13. Flu
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