产品描述
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.
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.
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
仅用于生命科学研究。不可用于诊断。
Quality
Function tested in the DIG RNA Labeling Kit and in the DIG Nucleic Acid Detection Kit.
Specificity
Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0).
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[3]
• Southern blots
• Dot blots
• Plaque or colony lifts
• RNase protection experiments
• Chromosomes, cells, and tissue sections in situ[1][2]
性质
