Biotin RNA Labeling Mix

Description

• General description: Convenient nucleotide mixture for the labeling of RNA with Biotin-16-UTP.
• Specificity: Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0).
• Application: Biotin RNA Labeling Mix has been used for RNA labeling with Biotin-16-UTP by in vitro transcription with SP6, T7, and T3 RNA polymerases.[1][2]Biotin-labeled RNA is also used in a variety of hybridization techniques: Southern blotsNorthern blots[3]Dot blotsPlaque or colony liftsRNase protection experimentsIn situ hybridizations[4]Microarray hybridizationsRNA pull down assay[1]
• Features and Benefits: Contents10x concentrated solution with: 10mM each ATP, CTP, GTP, 6.5mM UTP, 3.5mM Biotin-16-UTP, pH 7.5
• Quality: Function tested in combination with the DIG RNA Labeling Kit.
• Principle: Biotin-labeled, single-stranded RNA probes of defined length are generated by in vitro transcription. Biotin-16-UTP is incorporated by SP6, T7, and T3 RNA polymerases at approximately every 20 to 25th nucleotide of the transcript under the conditions described below. The Biotin RNA Labeling Mix is specifically designed for the use with Roche SP6,T7, and T3 RNA Polymerases, which are supplied with an optimized transcription buffer.
• Preparation Note: Assay Time: 135 minutesSample MaterialsLinearized plasmid DNA: The DNA to be transcribed is cloned into the polylinker site of an appropriate transcription vector which contains adjacent to the polylinker a promoter for SP6, T7 or T3 RNA polymerase. For the synthesis of ′run off′ transcripts the plasmid is linearized by a restriction enzyme. Restriction enzymes creating 5′-overhangs should be used; 3′ overhangs should be avoided. The linearized template DNA should be purified by phenolchloroform extraction and ethanol precipitation, to avoid RNase contamination. For ′run around′ transcription circular plasmid DNA is used.PCR product: PCR-fragments which contain RNA polymerase promoter sequences can also act as templates for transcription. Purification of the correct PCR fragment by gel electrophoresis prior to transcription is recommended.Labeling Efficiency :A standard labeling reaction with 1μg linear template DNA and an RNA polymerase produces approx. 10μg of full-length, biotin-labeled RNA. In a spot test, a combination of anti-biotin-AP and the chemiluminescence substrate CSPD can detect as little as 0.3pg of the biotin-labeled RNA.
• Other Notes: For life science research only. Not for use in diagnostic procedures.