Modification of 5 Phosphate Groups Using EDC

A kit designed specifically to perform 5 -phosphate labeling on DNA probes is available from Thermo Fisher. 

Weigh out 1.25mg of the carbodiimide EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide 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/pl or total of about 57-115.5 pg. Also immediately add 5 pi of 0.25 M cystamine in 0.1M imidazole, pH 6.0. Because EDC is labile in aqueous solutions, the addition of the oligo and cystamine/imidazole solutions should be done quickly. 

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

For reduction of the cystamine disulfides, add 20 pi of 1.0 M DTT and incubate at room temperature for 15 minutes. This will release 2-mercaptoethylamine from the cystamine modification site and create the free sulfhydryl on the 5 terminus of the oligonucleotide. 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mercaptoethylamine and creates a thiol group (Fig. 399). DNA probes labeled in this manner have been successfully conjugated with SPDP-activated alkaline phosphatase (Chapter 16, Section 1.5, and Section 2.4), maleimide-activated horseradish peroxidase (section 3.8.1), NHS-LC-biotin (Chapter 8, Section 3.1, and Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 8, Section 1.3, and Section 2.5). 

Add to the tube 7.5 xl of RNA or DNA containing a 5 phosphate group. The concentration of the oligonucleotide should be 7.5-15 nmol or total of about 

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