Member since: 2020
Organization: Ceva Biomune
Great kit to extract HMW DNA for long reads for next-generation sequencing using ONT.
Application Area: Generate long reads for Oxford Nanopore Tech next-generation sequencing
"Easy-to-follow steps to extract high molecular weight (HMW) DNA from both Gram-negative and Gram-positive bacteria. Extraction process is a little more difficult than spin column preps because you use glass beads to bind DNA. Sometimes you can extract such high concentrations of HMW DNA that it can take a couple days to weeks for DNA to relax and be ready to use for sequencing. Used in to extract DNA from unknown species and generated ONT sequencing reads over 250 Kbases in length! Was able to generate rough closed genomes on multiple unknown bacterial species."
For the extraction of HMW DNA from a variety of tissues, bacteria and other samples (yeast, insect, amphibian)
The Monarch HMW DNA Extraction Kit for Tissue provides a rapid and reliable process for extracting high molecular weight (HMW), intact genomic DNA from various tissues and bacteria, as well as other sample types including yeast, insect, and amphibian. The optimized extraction protocol for tissue utilizes pestle homogenization and proteinase K digestion with agitation for sample lysis, followed by a protein removal step and precipitation of the extracted DNA onto the surface of large glass beads. A slightly modified extraction protocol for bacteria utilizes lysozyme for the efficient lysis of the bacterial cell wall, prior to proteinase K digestion. DNA size ranges from 50- ≥500 kb for the standard protocols and into the Mb range when the lowest agitation speeds are used for processing soft organ tissues and bacteria. Purified DNA is recovered in high yield with excellent purity, including nearly complete removal of RNA. For tissue and bacteria, the processing time is 90 min. Purity ratios are typically 1.8-1.9 (260/280) for tissue and bacteria, 2.1-2.5 (260/230) for tissue, and 2.1-2.2 (260/230) for bacteria. Purified HMW DNA is suitable for a variety of downstream applications including long-read sequencing (Oxford Nanopore Technologies® and Pacific Biosciences®), optical mapping (Bionano Genomics®), and linked-read genome assembly.