For the most sensitive and reliable Nuclease Protection Assays (NPAs), probes should be full-length and purified. While precipitations and spin columns remove free nucleotides and DNase treatments can remove template DNA, these techniques do not allow qualitative analysis of the probe. Only by gel assessment can one reliably verify probe integrity. For both RNA and DNA probe preparations, gel purification, in a single step, removes free nucleotides, DNA template, buffer components, and enzymes, as well as prematurely terminated products. Prematurely terminated products can form during transcription if the polymerase encounters significant secondary structure or a homopolymeric stretch of one nucleotide, or if there is an inadequate amount of limiting nucleotide (typically the labeled nucleotide in the reaction). Such products will increase background bands and smearing on the Nuclease Protection Assay. With this in mind, we suggest that all probes used in Nuclease Protection Assays be gel purified.

Principle

The E.Z.N.A.® Poly-Gel RNA Extraction Kit is a simple system for recovering RNA probes from denaturing acrylamide gels. After the in vitro transcription reaction, the product is separated on a denaturing polyacrylamide gel to separate the DNA template, full-length RNA probe, any prematurely terminated products and free-nucleotides, by size. The gel is exposed to film, stained or UV shadowed. Full-length probe is identified and the band is excised from the gel. The probe is eluted by passive diffusion from the gel fragment and further purified using a HiBind® RNA spin column. The procedure can produce enough probes, ready for hybridization in just 1-4 hours.

Procedure

Applications

E.Z.N.A.^® Poly-Gel RNA Extraction Kit can be used for the following applications :

• Nuclease Protection Assays
• RT-PCR
• Ligation Reactions
• Hybridization Techniques