Purification of High Molecular Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap

<p><strong>Figure 3. Yield and purity of HMW DNA obtained from difficult samples using the method proposed in this study</strong>.</p> <p>(A) The proposed method extracts more HMW DNA from a complex soil sample than a commercial column purification kit. Shown is a negative image of an ethidium bromide-stained agarose gel. Lane 1: 1/10 aliquot of ~90 ng of HMW DNA isolated using the E.Z.N.A. soil DNA extraction kit from a soil sample containing ~1,5 &mu;g of total DNA (HMW DNA yield around 6%). Lane 2: ~100 ng of CTAB-extracted DNA from the same soil sample. ~10 &mu;g of this crude DNA preparation was used as input for HMW DNA purification using the proposed method. Lane 3: 1/100 aliquot of ~3 &mu;g of HMW DNA isolated using the proposed method from ~10 &mu;g of the CTAB-extracted DNA (HMW DNA yield around 30%).</p> <p>(B) The proposed method yields high-purity HMW DNA from a complex plant sample, as determined by agarose gel electrophoresis. Lane 1: molecular weight marker (GeneRuler DNA ladder, Thermo Fisher Scientific). Lane 2: crude nucleic acid preparation extracted with SDS/Proteinase K from <em>Zingeria trichopoda</em> leaves, which served as an input for HMW DNA purification using the proposed method. Lane 3: purified HMW DNA. Note the absence of low-molecular-weight nucleic acids and heavy covalent complexes in the purified sample.</p> <p>(C) Same as (B) except that crude, CTAB-extracted DNA from a complex soil sample was used as an input for HMW DNA purification. Note the absence of a continuous smear of fragmented DNA as well as heavy covalent complexes in the purified sample (lane 3). Molecular weight marker sizes are indicated in base pairs to the left of each panel.</p>