Description
The Ambion® MAXIscript® T3⁄T7 In Vitro Transcription Kit synthesizes RNA probes in just 10 minutes for use in ribonuclease protection assays, in situ hybridization, and blot hybridizations. Each kit contains sufficient reagents for 50 reactions of each enzyme.
• High yields of high specific activity, full-length RNA probes in just 10 minutes
• Ideal probe synthesis kit for nuclease protection assays, northern and Southern blots, and in situ hybridization
• Includes TURBO DNase™
• All kits contain quality-tested DNase⁄RNase-free reagents
The MAXIscript® In Vitro Transcription Kit synthesizes RNA probes with specific activities reaching 1 x 109 cpm⁄μg in just 10 minutes.MAXIscript® Kits are very efficient at synthesizing full-length probes, even at limiting nucleotide concentrations, making them the perfect companion to ribonuclease protection assays.
MAXIscript® Kits are also an excellent choice for the synthesis of nonisotopically labeled probes. Labeling can be accomplished post-transcriptionally by using Ambion's BrightStar® Psoralen-Biotin Labeling Kit, or during transcription by incorporating modified nucleotides into the reaction. MAXIscript® Kits can also be used for the synthesis of modest amounts of unlabeled RNA (2–6 μg⁄20 μl reaction) of up to 2 kb.
Nuclease protection assays (NPAs), including both ribonuclease
protection assays (RPAs) and S1 nuclease assays, are an extremely sensitive
method for the detection, quantitation and mapping of specific RNAs in
a complex mixture of total cellular RNA. The basis of NPAs is a solution
hybridization of a single-stranded, discrete sized antisense probe(s)
to an RNA sample (see Figure 1). The small volume solution hybridization
is far more efficient than more common membrane-based hybridization, and
can accommodate up to 100 µg of total or poly(A) RNA. After hybridization,
any remaining unhybridized probe and sample RNA are removed by digestion
with a mixture of nucleases. Then, in a single step reaction, the nucleases
are inactivated and the remaining probe:target hybrids are precipitated.
These products are separated on a denaturing polyacrylamide gel and are
visualized by autoradiography. If nonisotopic probes are used, samples
are visualized by transferring the gel to a membrane and performing secondary
detection.
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Northern analysis remains a
standard method for detection and quantitation of mRNA levels
despite the advent of powerful techniques, such as RT-PCR,
gene array analysis and nuclease protection assays. Northern
analysis provides a direct relative comparison of message
abundance between samples on a single membrane. It is the
preferred method for determining transcript size and for
detecting alternatively spliced transcripts.
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The MAXIscript®
in vitro Transcription Kit from Ambion was developed for the synthesis of highly ...
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