General description
Assay Time: 145minutes
Sample Materials
- Linearized
plasmid DNA
- PCR
product
Principle
The 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 Transcription
The 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.
Kit for the labeling of RNA with digoxigenin-UTP by in
vitro transcription with SP6 and T7 polymerases. By this method,
single-stranded RNA probes of known length are produced, which can be used in a
variety of hybridization techniques.
We are committed to bringing you Greener Alternative
Products, which adhere to one or more of The 12 Principles of Greener
Chemistry. This product is designed as a safer chemical. The DIG System
was established as a sensitive and cost-effective alternative to using
radioactivity for the labeling and detection of nucleic acids. There are many
available publications that prove the versatility of the DIG System, so use of
radio-labeling is no longer the only option for labeling of DNA for hybridization.
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
blots
- In
situ hybridizations[2][3][4]
- Plaque
or colony lifts
- RNase
protection experiments
Due 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.
Biochem/physiol Actions
Sensitivity and Specificity
DIG-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).
Packaging
1 kit containing 12 components.
Analysis Note
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.
