Cy5-UTP sodium (10 mM in water)

Cy5-UTP sodium can replace UTP as the substrate for T7 RNA polymerase and can be used for in vitro transcription to generate labeled probes. With the participation of Cy5-UTP sodium, the final assembled mRNA will emit orange fluorescence. Moreover, the cy5-labeled mRNA can be easily observed under ultraviolet light after gel electrophoresis without the need for further staining^[1][2].

Protocol (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
1. Prepare reagents
Apart from this product, prepare the following:
(1) T7 RNA polymerase (HY-E70090)
(2) Reaction buffer: 0.1 M sodium bicarbonate buffer (pH: 7-8.5)
(3) 50 mM UTP stock solution
(4) ATP/CTP/GTP stock solutions, with each ribonucleotide at 25 mM.
Note: Thaw the above reagents (except enzymes) on ice.
2. Experimental Procedures
(1) Add the following to an RNA-free Eppendorf tube: 2 μL reaction buffer, 6 μL ATP/CTP/GTP stock solution, 2 μL UTP stock solution, 6 μL Cy5-UTP solution, and 4 μL sterile water; mix thoroughly.
(2) Add the DNA template and T7 RNA polymerase; gently stir to mix evenly.
(3) Incubate in the dark at 37°C for 30 minutes to several hours (depending on the yield).
(4) Add 1 μL of Recombinant DNase I (RNase-free) (HY-108882A) to remove the template DNA. Mix gently and centrifuge. Then incubate at 37 °C for an additional 10 minutes.
3. Detection: The fluorescently labeled RNA can be directly observed under ultraviolet light after gel electrophoresis, or it can be examined using a fluorescence microscope.
Note: (1) Only water and consumables without RNase should be used.
(2) The ratio of Cy5-UTP to natural UTP usually needs to be adjusted to strike a balance between optimal labeling efficiency and transcriptional yield.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.