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

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3-Linker Ligation and Size Selection by SDS-PAGE

(Protocol summary only for purposes of this preview site)

In this protocol, coimmunoprecipitated RNA tags are treated with alkaline phosphatase to remove the 3 phosphate remaining after RNase digestion. Dephosphorylation prevents intramolecular circularization of RNA during subsequent ligation to the linker. An RNA linker, blocked with puromycin at its 3 end to prevent linkerlinker multimerization, is ligated to the 3 end of the RNA tag. Removal of free linker is accomplished by performing the ligation while the RNABP:RNA complex is associated, via antibody, to protein A Dynabeads, allowing thorough washing and linker removal. Additional purification is achieved by SDS-PAGE and transfer of the size-selected RNABP:RNA complexes to nitrocellulose.


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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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