Description
General description
Sample Material
DNA inserted into the transcription vectors pSPT18 or pSPT19.The template DNA must be linearized with a suitable restriction enzyme before the transcription reaction to obtain transcripts of a defined length. Using intact plasmid DNA as template for transcription will result in heterogeneous transcripts of multiple plasmid lengths.
Application
Convenient kit for radioactive or nonradioactive labeling of RNA by the in vitro transcription with SP6 and T7 polymerase. The kit can also be used for "cold" transcription assays. By the in vitro transcription method single-stranded RNA probes of known length are produced, which can be used in a variety of hybridization techniques.
For in vitro transcription of DNA sequences cloned downstream of the SP6 or T7 promoter. Homogeneously labeled RNA can be synthesized with high efficiency (60 - 70% incorporation) using either radioactively (e.g.,³²P, ³H, ³⁵S) labeled or nonradioactively (e.g., digoxigenin or biotin) labeled ribonucleotides. Labeled transcripts lend themselves to all DNA and RNA hybridization techniques and are also used for genomic sequencing and S1 nuclease studies. Large amounts of highly pure RNA can be synthesized using the SP6/T7 system. These transcripts are used for studies on RNA-processing systems. Synthesized RNA can be translated in vitro, or in vivo after injection into oocytes. The transcription of defined mRNA can be inhibited by the introduction of "antisense"-RNA. The efficiency of in vivo translation of synthesized mRNA can be increased significantly by the introduction of a cap structure.
Biochem/physiol Actions
Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0) and/or heating to 65 °C for 10 minutes.
DNA is inserted into the polylinker site of the transcription vectors pSPT18 or pSPT19; these two vectors differ only in the orientation of their polylinker regions. The promoters for SP6 and T7 RNA polymerases are located on either side of the polylinker. SP6 and T7 RNA polymerases specifically transcribe DNA sequences downstream of the SP6 or T7 promoters, respectively. Cloned inserts within the polylinker region are transcribed from either promoter. The first DNA strand may be transcribed with SP6 RNA polymerase and the opposite strand using T7 RNA polymerase. It is also possible to transcribe the first and opposite strands by inserting the same DNA into both pSPT18 and pSPT19 in opposite orientations and transcribing with only one of the RNA polymerases. SP6 and T7 RNA polymerase use the cloned DNA as template and synthesize complementary RNA in the presence of Mg2+ and ribonucleoside triphosphates. Spermidine stimulates enzyme activity. Specifically labeled transcripts are obtained when using radioactively (e.g., 32P, 3H, 35S) or nonradioactively (e.g., digoxigenin or biotin) labeled ribonucleotide triphosphates.
Packaging
1 kit containing 12 components.
Preparation Note
Working solution: Standard Labeling Assay
ATP, GTP, UTP mixture
Prepare the ATP, GTP, UTP mixture by making a 1:1:1 mixture of solution 4, solution 6, and solution 7.
Transcription Assay with digoxigenin-11-UTP
ATP, GTP, CTP mixture 1
Mix 1:1:1 of ATP (vial 4), CTP (vial 5), and GTP (vial 6).
UTP/DIG-11-UTP mixture
Mix 1:1 digoxigenin-11-UTP (6 mM) with UTP (vial 7) and add as one part to mixture 1.
"Cold" Transcription
ATP, GTP, CTP, UTP mixture
Prepare this mix by combining solutions in vials 4, 5, 6 and 7 at a ratio of 1111.
Other Notes
For life science research only. Not for use in diagnostic procedures.
