Alkylphosphates with Bis-Hydrazide Compounds

Phosphoramidate Linkage with Terminal Hydrazide Group 

Weigh out 1.25 mg of the carbodiimide EDC hydrochloride (Thermo Fisher) into a microfuge tube. 

Add to the tube 7.5 pi of RNA or DNA containing a 5 -phosphate group. The concentration of the oligonucleotide should be 7.5-15 nmol or total of about 57-115.5 pg. Also, immediately add 5 pi of 0.25 M adipic acid dihydrazide or carbohydrazide dissolved in 0.1 M imidazole, pH 6.0. Because EDC is labile in aqueous solutions, the addition of the oligo and fezs-hydrazide/imidazole solutions should occur quickly. 

Mix by vortexing, then place the tube in a microcentrifuge and spin for 5 minutes at maximal rpm. 

Add an additional 20 pi of 0.1M imidazole, pH 6.0. Mix and react for at least 2 hours at room temperature. The additional buffer prevents pH drift during the carbodiimide reaction. 

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