BIOTIN RNA LABELING MIX 10X

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.
Biotin-labeled RNA is also used in a variety of hybridization techniques:

• Southern blots
• Northern blots
• Dot blots
• Plaque or colony lifts
• RNase protection experiments
• In situ hybridizations
• Microarray hybridizations
• RNA pull down assay

Features and Benefits

Contents
10x 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 minutes

Sample Materials

• Linearized 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.