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Fluorescein-12-dUTP Protocol

Product No. 11373242910

Protocol

Labeling of RNA at the 3′-end with Fluorescein-12-UTP using Poly(A) Polymerase:
This is not possible, because Poly(A) Polymerase catalyzes the incorporation of adenine residues only into the 3′-termini of single-stranded RNA. The enzyme incorporates adenine residues and uses ATP as its only substrate.

Instead, Terminal Transferase can be used to tail RNA molecules with labeled-dUTP. The conditions for adding a tail of modified nucleotides to RNA are similar to those described for DNA:
Incubate 1 μg RNA with 25 U Terminal Transferase and 1 U Protector RNase Inhibitor in 200mM potassium cacodylate, 25 mM Tris-HCl (pH 6.6), 0.25 mg/ml BAS, 5 mM CoCl2, and 0.5 mM labeled dUTP (DIG-, biotin-, or fluorescein-dUTP) in a final volume of 30 μl for 30 min at +37 °C. For longer tails, a nucleotide mixture containing 0.45 mM dATP and 0.05 mM labeled dUTP is used. (from Rosemeyer et al., Nonradioactive 3 '-End-Labeling of RNA Molecules of Different Lengths by Terminal Transferase. Anal. Biochem. 224 (1995), pp. 446-449.)

Materials

     
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