11175025910

Roche

DIG RNA Labeling Kit (SP6/T7)

greener alternative

usage

sufficient for 2 x 10 labeling reactions

Quality Level

manufacturer/tradename

Roche

greener alternative product characteristics

Designing Safer Chemicals
Learn more about the Principles of Green Chemistry.

shipped in

dry ice

storage temp.

−20°C

General description

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.
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.

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).

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.

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.

Kit Components Only

Product No.
Description

  • pSPT18 DNA 0.25 mg/ml

  • pSPT19 DNA 0.25 mg/ml

  • Control DNA 1, pSPT18-Neo, cleaved with Pvu II 0.25 mg/ml

  • Control DNA 2, pSPT19-Neo, cleaved with Pvu II 0.25 mg/ml

  • DIG-labeled Control RNA, DIG-labeled "antisense" neo RNA 100 ng/µl

  • Unlabeled Control RNA, neo poly (A) "sense" RNA 200 µg/ml

  • NTP Labeling Mixture 10x concentrated

  • Transcription Buffer 10x concentrated

  • DNase I, RNase-free 10 U/µl

  • Protector RNase Inhibitor 20 U/µl

  • SP6 RNA Polymerase 20 U/µl

  • T7 RNA Polymerase 20 U/µl

See All (12)

pictograms

Exclamation mark

signalword

Warning

hazcat

Acute Tox. 4 Oral - Eye Irrit. 2

storage_class_code

12 - Non Combustible Liquids

WGK Germany

WGK 2

Flash Point F

does not flash

Flash Point C

does not flash

Certificate of Analysis

Certificate of Origin

Maria Lynn Spletter et al.
Neural development, 2, 14-14 (2007-07-20)
Precise connections of neural circuits can be specified by genetic programming. In the Drosophila olfactory system, projection neurons (PNs) send dendrites to single glomeruli in the antenna lobe (AL) based upon lineage and birth order and send axons with stereotyped...
Parvaneh Alizadeh et al.
Experimental eye research, 83(3), 679-687 (2006-05-11)
Cystatin C is the major inhibitor of the cysteine cathepsins. Polymorphisms in the cystatin C gene have recently been associated with the risk of developing Age-related Macular Degeneration (AMD). Oxidative stress is also thought to play a key role in...
Nathan Luebbering et al.
PloS one, 8(10), e76775-e76775 (2013-10-23)
The DYRKs (dual-specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that are associated with a number of neurological disorders, but whose biological targets are poorly understood. Drosophila encodes three Dyrks: minibrain/Dyrk1A, DmDyrk2, and DmDyrk3. Here we describe...
Yu Liang et al.
Molecular medicine reports, 5(4), 1087-1091 (2012-01-17)
Schistosoma japonicum (S. japonicum) is an extremely harmful pathogen, which infects humans and causes severe public health problems. To date, no effective therapeutic drugs for this pathogen are available. In this study, we designed and constructed three hammerhead ribozymes targeting...
Anna Karlgren et al.
Journal of visualized experiments : JoVE, (26)(26), doi:10-doi:10 (2009-04-21)
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented...
Articles
Digoxigenin (DIG) labeling methods and kits for DNA and RNA DIG probes, random primed DNA labeling, nick translation labeling, 5’ and 3’ oligonucleotide end-labeling.
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Protocols
Determine the labeling efficiency in terms of μg (expected yield of a standard labeling reaction is 20 μg of DIG labeled RNA per μg linearized template DNA after the DIG RNA labeling reaction).
Read More

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