Description
General description
Labeling efficiency: Approximately 10μg of full-length digoxigenin-labeled RNA is transcribed from 1μg linear template DNA.
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 phenol chloroform 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 be used as templates for transcription. Purification of the PCR fragment by HighPure column purification prior to transcription is recommended.
DIG-labeled, single-stranded RNA probes of defined length are generated by in vitro transcription. DIG-11-UTP is incorporated by SP6, T7, and T3 RNA polymerases at approximately every 20 to 25th nucleotide of the transcript under standard conditions. The DIG RNA Labeling Mix is specifically designed for the use with SP6, T7, and T3 RNA polymerases, which are supplied with an optimized transcription buffer.
Convenient nucleotide mixture for the labeling of RNA with Digoxigenin-11-UTP.
Contents
10x solution with: 10 mM ATP, CTP, GTP (each), 6.5 mM UTP, 3.5 mM DIG-11-UTP.
Application
RNA labeling with Digoxigenin-11-UTP by in vitro transcription with SP6, T7, and T3 RNA polymerases. DIG-labeled RNA is used in a variety of hybridization techniques:
Northern blots
Southern blots
Dot blots
Plaque or colony lifts
RNase protection experiments
Chromosomes, cells, and tissue sections in situ
Biochem/physiol Actions
Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0).
Features and Benefits
The DIG RNA Labeling Mix is especially designed for the use with SP6,T7 and T3 RNA polymerases from Roche which are supplied with an optimized transcription buffer.
Analysis Note
Function tested in the DIG RNA Labeling Kit and in the DIG Nucleic Acid Detection Kit.
Other Notes
For life science research only. Not for use in diagnostic procedures.
