Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Analysis of Small RNAs by Northern Hybridization

(Protocol summary only for purposes of this preview site)

This protocol describes how to use northern hybridization to detect 15150-nucleotide small RNAs (Fig. 1). Total RNA is fractionated by electrophoresis through a denaturing polyacrylamide gel and then transferred to a nylon membrane by semidry electroblotting. After UV-cross-linking the RNA to the membrane, hybridization is performed in Church buffer using a 32P-radiolabeled oligonucleotide probe followed by PhosphorImager analysis. The use of StarFire probes allows a substantial increase in the specific radioactivity of the hybridization probe. StarFire probes contain two domains. The target-specific domain at the 5 end hybridizes to the small RNA of interest. The universal template-binding domain at the 3 end is used to bind a universal template oligonucleotide that carries 10 deoxythymidines at its 5 end, providing a template for DNA polymerase to incorporate 10 [-32P]deoxyadenosines at the 3 end of the StarFire probe. (See the information panel StarFire Probes.)


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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