DIG RNA Labeling Kit (SP6/T7)

Description

• General description: Assay Time: 145minutesSample MaterialsLinearized plasmid DNAPCR product PrincipleThe DIG RNA Labeling Kit produces DIG-labeled, single-stranded RNA probes of known length. Either SP6 or T7 RNA polymerase transcribes these probes in vitro from template DNA (in the presence of digoxigenin-UTP).RNA Labeling by in vitro TranscriptionThe DNA to be transcribed is cloned into the polylinker site of appropriate transcription vectors (e.g., pSPT 18 or 19), which contain promoters for SP6 and T7 RNA polymerases. Adjacent template DNA is linearized at a suitable site and the RNA polymerases are used to produce "run off" transcripts. DIG-UTP is incorporated into the transcript. Every 20 to 25th nucleotide of the newly synthesized RNA is a DIG-UTP. Since the nucleotide concentration does not become limiting in the standard transcription reaction, this reaction can generate large amounts of labeled RNA.
• Specificity: Sensitivity and SpecificityDIG-labeled RNA probes can detect single-copy genes in as little as 1 μg of mammalian DNA under the following assay conditions: The hybridization mix contains 20 to 100 ng labeled probe/ ml, and the bound probe is detected with anti-DIG-AP and visualized with the chemiluminescent substrate CDP-Star.Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0).
• Application: For RNA labeling with DIG-11-UTP by in vitro transcription with SP6 and T7 RNA Polymerases. DIG-labeled "run off" transcripts are synthesized with high efficiency and can be used in a variety of hybridization techniques: Northern blots[1]Southern blotsIn situ hybridizations[2][3][4]Plaque or colony liftsRNase protection experimentsDue to highly specific and sensitive detection systems, DIG-labeled probes can be used for single-copy gene detection in 1μg total human DNA.Note: Since the linkage between DIG and UTP is resistant to alkali, DIG-labeled RNA can be fragmented by alkaline treatment. Slightly reducing the size of the DIG-labeled RNA probe may make it more suitable for certain applications in in situ hybridization.
• Packaging: 1 kit containing 12 components.
• Quality: Test principle: The DNA template to be transcribed is cloned into the polylinker site of appropriate transcription vectors, which contain promoters for SP6 and/or T7 RNA Polymerases. After linearization at a suitable site, RNA is transcribed in the presence of DIG-UTP. Under standard conditions, approximately 10μg of full-length DIG-labeled RNA is transcribed from 1μg template.
• Other Notes: For life science research only. Not for use in diagnostic procedures.