Choosing an Agitation Speed During Lysis with the Monarch HMW DNA Extraction Kits

The Monarch HMW DNA Extraction Kits enable users to tune the size of extracted HMW by varying the agitation speed used during lysis. Higher agitation speeds reduce overall size, as increasing agitation speeds fragments the DNA. In general, fresh samples have more intact DNA and will require higher agitation rates to shear. At 2,000 rpm, the maximal fragment length obtained from cells and blood samples will be ~250–300 kb, with the majority of DNA between 100–200 kb. For the standard ligation-based Oxford Nanopore Technologies (ONT) sequencing protocol, agitation at 2000 rpm is recommended. At 300 rpm or with no shaking, maximal fragment length, in the Mb range, will be obtained (XL DNA). These samples will be highly viscous and difficult to process. 

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 µl 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. 


Use of varying agitation speeds during lysis produces tunable fragment length of extracted HMW genomic DNA 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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