Isopropanol DNA Precipitation - Best for DNA Concentration v1

Protocol to precipitate extracted DNA from an aqueous solution to increase concentration or resolubilize in a different storage buffer. *Use isopropanol DNA precipitation if your DNA is suspended in a very large volume, if your DNA concentration is low, or you are trying to concentrate large molecular weight DNA fragments and remove smaller fragments. *Use ethanol DNA precipitation if you are trying to remove salt contamination or precipitate small DNA fragments. Additional Resources: New England Biolabs, "DNA Precipitation: Ethanol vs. Isopropanol". June 23, 2015 Green, Michael R. and Joseph Sambrook, "Precipitation of DNA with Isopropanol". doi:10.1101/pdb.prot093385Cold Spring Harb Protoc2017. Qiagen, "How can I precipitate genomic DNA using isopropanol?".

Mapping the Location of Nucleosomes in Simian Virus 40 Chromatin Using the New England Biolabs FS Library Prep Kit

The location of nucleosomes in chromatin significantly impacts many biological processes including DNA replication, repair and gene expression. A number of techniques have been developed for mapping nucleosome locations in chromatin including MN-Seq (micrococcal nuclease digestion followed by next generation sequencing), ATAC-Seq (Tagamet chromatin fragmentation followed by next generation sequencing), and ChIP-Seq (chromatin immunoprecipitation and fragmentation followed by next generation sequencing). All of these techniques have been successfully used, but each with its own limitations. Recently, New England Biolabs has marketed a new kit, the NEBNext UltraII FS Library Prep kit, for preparing libraries for next generation sequencing from purified genomic DNA. This kit is based on a novel proprietary DNA fragmentation procedure which appears to cleave DNA that is not bound by proteins. Because DNA is fragmented directly in the FS kit, we tested whether the kit might also be useful for mapping the location of nucleosomes in chromatin. Using Simian Virus 40 (SV40) chromatin isolated at different times in an infection, we have compared nucleosome mapping using the NEB FS kit (FS-Seq) to MN-Seq, ATAC-Seq, and ChIP-Seq. Mapping nucleosomes using FS-Seq generated nucleosome profiles similar to those generated by ATAC-Seq and ChIP-Seq in regulatory regions of the SV40 genome. We conclude that FS-Seq is a simple, robust, cost-effective procedure for mapping nucleosomes in SV40 chromatin that should be useful for other forms of chromatin as well. We also present evidence that the FS kit may be useful for mapping the location of transcription factors in chromatin when sequencing reads between 75 and 99 base pairs in size are analyzed.

Report on Overcoming the Poor Quality of ApaI Pulsotypes with a Short Review on PFGE for Listeria monocytogenes

Since listeriosis, caused by Listeria monocytogenes, is one of the important concerns of public health in Europe related to foodborne zoonoses, an efficient protocol for isolate typing is necessary when performing epidemiological studies. Three standardized PFGE protocols available for L. monocytogenes were briefly reviewed. Since observing a poor-quality of ApaI pulsotypes in our laboratory, enzymes from three different manufacturers were compared. The obtained pulsotypes showed that restriction digestion with ApaI from New England BioLabs should be complemented with a subsequent overnight incubation of PFGE plugs in TE buffer for better performance.