Computational protocol: An optimized two-finger archive for ZFN-mediated gene targeting

Similar protocols

Protocol publication

[…] The selections for 3F and 4F ZFAs were performed as previously described. 1–5×107 selection strain cells transformed with the 1352-omega-UV2 ZFA expression plasmid and the 28 bp pH3U3 library plasmid were plated on NM minimal medium selective plates lacking uracil and containing 3-AT (2.5, 5 or 10 mM) as the competitor and grown at 37 °C for 36–72 hours. The number of surviving bacterial colonies on each plate was estimated and then these colonies were pooled and the population of recovered DNA sequences was determined via Illumina sequencing. Unique sequences were ranked based on the number of recovered reads. From this list an overrepresented sequence motif was determined with MEME using as input the number of unique sequences from the top of the list that correspond to the estimated number of colonies on the selection plate (typically >1000). The aligned sequences were then used to generate Sequence logos using Weblogo. […]

Pipeline specifications

Software tools MEME, WebLogo
Application Genome data visualization
Organisms Danio rerio
Chemicals Zinc