User Manual
Contents
1. 100 rxn size Tailing Enzyme 100 pL 10X Reaction Buffer 200 uL 10 mM ATP Solution 200 uL Protocol The following protocol is designed as a general guideline for generating a poly A tail length of 100 bases Customizing reaction to reach desired tailing length could be reached by adjusting such as enzyme concentra 1 650 872 0245 www mclab com 1 tion and or incubation time 1 Set up reaction in a PCR tube on ice as below Component Quantity Note 10x Reaction Buffer 2 uL 1x Nuclease free water To 20 uL 10mM ATP 2 uL 1mM Tailing Enzyme Mix 1 uL RNA Template x uL 1 10ug For multiple reactions master mix should be made with 5 extra reagents to reduce pipette error 2 Gently mix thoroughly and then centrifuge briefly 3 Incubate the tube at 37 C for 20 minutes 4 Stop reaction by appropriate methods according downstream applications such as 20 C storage EDTA addition or through common purification process References 1 Drummond D R et al 1985 J Cell Biol 100 1148 2 Galili G et al 1988 J Biol Chem 263 5764 3 Lingner J and Keller W 1993 Nucleic Acids Res 21 2917 2 lt MCLAB2. lt gt MCLAB Molecular Cloning Laboratories User Manual Version 1 0 Revision Date 08 23 2013 Product name RNA Poly A Tailing Kit Cat RPTK 100 RPTK 200 Description The RNA Poly A Tailing Kit provides high pure and active enzyme and other reagents for quickly and easily adding a poly A tail to the 3 end of any RNA MCLAB newly engineered Poly A Polymerase uses ATP as a substrate for template independent addition of adenosine monophosphate to the 3 hydroxyl termini of RNA molecules Cloned Poly A Polymerase is encoded by an E coli gene and overexpressed in the host strain Applications Addition of a poly A tail to an RNA molecule or a mixture of RNA molecules in order to provide a priming site for synthesis of first strand cDNA using a primer with poly dT on its 3 end Addition of a poly A tail to RNA synthesized in vitro in order to increase the stability of the RNA and en hance its ability to be translated in vivo after transfection or microinjection into eukaryotic cells Synthesis of polyadenylated RNA for nucleic acid amplification methods or gene expression studies 3 end labeling of RNA or quantitate mRNA Recommended Storage Conditions 20 C List of Components RNA Poly A Tailing Kit is supplied for 20 or 100 tailing reactions of 20 uL each RNA Poly A Tailing Kit 20 rxn size Tailing Enzyme Mix 20 pL 10X Reaction Buffer 200 uL 10 mM ATP Solution 40 uL RNA Poly A Tailing Kit
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