For the extraction of HMW DNA from a variety of tissues, bacteria and other samples (yeast, insect, amphibian)
Isolate DNA into the Mb size range from soft organ tissues and bacteria
For some samples, the size of isolated DNA can be tuned by adjusting the agitation speed during lysis
Fast, user-friendly workflows (90 minutes for tissues and bacteria)
Obtain excellent yields of highly-pure and intact DNA
Rapid and efficient elution
Ideal for long-read sequencing applications
Includes RNase A and Proteinase K, as well as microtube pestles for convenient sample homogenization
Featured Resources:
See how the Monarch HMW DNA workflow significantly reduced workflow time in the Cas9 targeted sequencing approach in our joint Technical Note with The Jackson Laboratory.
Check out our publication in Biotechniques outlining a simple approach to accurate quantitation of ultra high molecular weight (UHMW) DNA.
Review our technical note examining the use and comparison of a novel method to isolate genomic DNA from bacteriophages.
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.
Figure 1: Workflow for isolation of HMW DNA from Tissue using the Monarch HMW DNA Extraction Kit
Figure 2: The Monarch HMW DNA Extraction Kit for Tissue efficiently purifies high-quality, HMW DNA from a variety of sample types
HMW genomic DNA extracted with the Monarch HMW DNA Extraction Kit for Tissue. 10 mg frozen rat kidney, 10 mg fresh mouse liver, 20 mg frozen mouse brain, 20 mg fresh mouse muscle, 2 x 109 frozen E. coli cells, 2 x 108 frozen B. cereus cells, 4 mg fresh X. laevis, 3.8 x 108 fresh S. cerevisiae cells and 15 mg frozen A. aegypti were used as inputs for preps. Preps were performed according to the kit instructions with sample agitation at 2000 rpm. A modified workflow was used to process S. cerevisiae preps. 500 ng of DNA from each sample prep was resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5-94 seconds on a BioRad CHEF-DR III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder (NEB #N0341) was used as a molecular weight standard.
Figure 3: Use of varying agitation speeds during lysis produces tunable fragment length for HMW genomic DNA extracted from soft organ tissues and bacteria
HMW genomic DNA from mouse kidney (10 mg, Figure 3A) and E. coli NEB10 beta (1 x 109 cells, Figure 3B) was purified using the Monarch HMW DNA Extraction Kit for Tissue. Samples were agitated at the indicated speed during the lysis step to control the fragmentation of the DNA. 300 ng (kidney) and 500 ng (E. coli) of DNA for each sample was resolved by PFGE (settings: switch time 0.5 to 94 sec; run time 20 hours at 6 V/cm, angle 120°, temperature 13°C on the BioRad CHEF-DR III System). Lambda PFG Ladder (NEB #N0341) was used as molecular weight standard. Yield, purity ratios and DIN values (kidney) of the individual preps are shown in the accompanying tables
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