Biotin-16-UTP

This plan only provides a guide, please modify it to meet your specific needs.

1. Solution preparation

(1) Stock solution: This product is provided in 10mM stock liquid form.

Note: After the unused stock solution is aliquoted, store it at -20°C or -80°C in the dark to avoid repeated freezing and thawing.

(2) Working solution: Dilute the stock solution with the experimental buffer to prepare a working concentration of 0.5mM.

Note: Please adjust the working solution concentration according to the actual situation and prepare it before use.

2. Preparation of RNA probe with Biotin-16-UTP^[1](from the literature, for reference only)

(1) Template DNA preparation: In a 0.5mL test tube, add 1μg purified template DNA or 4μL control DNA to sterile, RNase-free diethyl pyrocarbonate (DEPC) water, and the total sample volume is 13μL.

(2) Preparation of in vitro transcription reaction system: For each sample, add 2μL 10× deoxynucleoside triphosphate mixture, 2μL 10× transcription buffer, 1μL Protector RNase inhibitor and 2μL RNA polymerase SP6 or RNA polymerase T7.

(3) Labeling reaction: Add Biotin-16-UTP (final concentration is usually 0.5mM) and incubate at 37°C for 2h to prepare RNA probe.

(4) Termination reaction: Add 2mL RNase-free-DNase I to each tube to remove template DNA and incubate at 37°C for 10min. Add 2mL 0.2M EDTA (pH 8.0) to each tube to terminate the DNase I reaction.

(5) Isolation of RNA probe: Isolate RNA probe by lithium chloride precipitation and resuspend in 25μL DEPC water.

References:
[1] Bharati B K, Swetha R K, Chatterji D. Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover[J]. Gene, 2013, 528(2): 99-108.