The Monarch HMW DNA Extraction Kit for Cells & Blood provides a rapid and reliable process for extracting high molecular weight (HMW), intact genomic DNA from cultured cells and whole blood. Utilizing an optimized process that combines gentle cell lysis with a tunable fragment length generation followed by precipitation of the extracted DNA onto the surface of large glass beads, the prep proceeds rapidly and utilizes standard laboratory equipment. DNA size ranges from 50-250 kb for the standard protocol and into the Mb range when the lowest agitation speeds are used. Purified DNA is recovered in high yield with excellent purity, including nearly complete removal of RNA. For cells, the process time is only 30 min, while blood samples require erythrocyte lysis and are processed in 60 min. Purity ratios of 1.80-1.90 and 2.2-2.5 (A260/A280 and A260/A230, respectively), are easily and reproducibly achievable, and 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 cells using the Monarch HMW DNA Extraction Kit
Figure 2: Workflow for isolation of HMW DNA from blood using the Monarch HMW DNA Extraction Kit
Figure 3: Reproducible Extraction of HMW DNA from Cells and Blood with the Monarch HMW DNA Extraction Kit
DNA extracted with Monarch HMW DNA Extraction Kit for Cells & Blood. 1 x 106 fresh HEK293 cells and 500 µl fresh human blood were used as inputs and for preps performed according to the kit instructions using the agitation speed indicated above the gel lanes. 500 ng of DNA from the replicates 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 molecular weight standard.
Figure 4: Use of varying agitation speeds during lysis produces tunable fragment length of extracted HMW genomic DNA from cells and blood
Preps were performed on duplicate aliquots of 1 x 106 HEK 293 cells and 500 ìl fresh human blood. Samples were agitated at the indicated speed during the lysis step to control the fragmentation of the DNA. Equal amounts of DNA from the replicates (cells: 500 ng; blood: 650 ng) were 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 and Lambda DNA-Hind III Digest (NEB #N0341 and #N3012) were used as molecular weight standards. Yield, purity ratios and DINs of the individual preps are shown in the accompanying tables.
Figure 5: Linear correlation between DNA yield and input for cell and blood samples
Summarized yield data for HMW DNA preps are shown carried out at 2,000 rpm during lysis, using HEK293 cultured cells and fresh human blood samples from different donors as input material in the corresponding protocols. The starting materials were diluted to 5 different concentrations to cover the entire recommended input range. Cell samples ≤ 5x105 cells and blood samples <500 µl were purified using the recommended volumes for low input samples. Obtained yields show a high degree of linearity over the displayed input range.
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