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
??Southern blots
??In situ?hybridizations
??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.

General description

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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 vitrofrom 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.

Sigma Life Science is 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.

Other Notes

For life science research only. Not for use in diagnostic procedures.

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.

Specificity

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).