pET100 Directional TOPO

Retinal Transcriptome Profiling by Directional Next-Generation Sequencing Using 100 ng of Total RNA

profiling. However, its usage will expand dramatically if one starts with low amount of RNA and obtains transcript directionality during the analysis. Here, we describe a detailed protocol for the creation of a directional RNA-seq library from 100 ng

Fill-in Labeling of DNA : directional end label

dNTP Stocks Thaw100 mM stocks (NEB or Boehringer Mannheim) on ice and dilute 10-fold in Q. store small (10-20 m l) aliquotes at -80 degrees and thaw on ice just prior to use 10 X Klenow Buffer 0.5 M Tris 7.5 500 0.1 M MgCl2 m l 1 M Tris 7.5 2 100

Generation of uni-directional nested deletions in plasmid DNA

. 7) Remove a 2μl aliquot of the pre-incubated deletion mix into the tube labelled t=0 and hold on ice. Return the deletion mix to the water bath at the correct temperature, add 1μl ExoIII (~100U), mix gently by pipetting up and down and return to the water