Description
The Ambion® MAXIscript® 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 100 reactions.
• 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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