Principle / Procedure

The NucleoBond® HMW DNA enables isolation of high quality DNA from gram positive and gram negative bacteria, yeast, plants, insects, mammalian tissue, and blood. The anion exchange column included in the kit ensures high purity of the isolated DNA regardless of the starting material and lysis method.

In addition, the midi format of the column allows for the use of up to 1.5 g of plant material or 300 mg of mammalian tissue in each prep. This makes NucleoBond® HMW DNA ideal for the commonly used long-read sequencing technologies such as SMRT and Nanopore as well as long-range short-read methods such as Next GEM (10X Genomics).

NucleoBond® HMW DNA is compatible with several different methods for sample lysis to accommodate different lab setups and user priorities. The kit can be combined with NucleoSpin® Bead Tubes, NucleoSnap® Finishers or NucleoSpin® Finishers for fast sample homogenization and DNA concentrations/desalting, respectively. Alternatively, various commercially available enzymes can be used for prelysis and combined with DNA precipitation for optimal DNA integrity. Proteinase K, which is required for enzymatic lysis of protein-rich samples as well as for inactivation of nucleases is included in the kit thus ensuring high quality isolations for animal cells and tissues.

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Universal core procedure The NucleoBond® HMW DNA kit allows for diverse, sample specific lysis protocols. Following the lysis step, the lysate is loaded onto the NucleoBond® HMW DNA Column. The lysate is cleared in parallel with the binding thanks to the specially designed, disposable column filters. Following the sample binding step and discarding of the filter, a wash procedure ensures efficient removal of inhibitors and contaminants while avoiding any shearing of the HMW DNA. After elution, the DNA is desalted and concentrated following one of the several procedures that are compatible with the kit.

Custom protocol optimization Depending on priorities, there are several options for workflow optimization. For high integrity and length of DNA, it is recommended to perform enzymatic prelysis of bacterial and yeast samples. For a faster procedure with intermediate DNA integrity, many samples can be homogenized in bead tubes (e.g., NucleoSpin® Bead Tubes) prior to lysis. Some samples, such as plant tissues, can also be homogenized with a mortar and pestle. Following the wash and elution steps, the user can choose a faster procedure for DNA desalting on a vacuum manifold (NucleoSnap® Finishers) or using a centrifuge (NucleoSpin® Finishers ). Alternatively, for optimal DNA integrity, isopropanol precipitation is recommended.

Application Data

Large fragments of DNA DNA was purified from Brassica sp. by mechanical lysis. The purified DNA was then analyzed on a Femto Pulse System. Fragments up to 200000 bp could be detected with a peak at 117218 bp, demonstrating the ability of the NucleoBond® HMW DNA kit to extract and purify high molecular weight DNA.
Optimal procedure for longer reads DNA was purified from Brassica sp. by mechanical lysis. The purified DNA was sequenced on a MinION device (Oxford Nanopore). More than 1600 reads longer than 50 kbp, inlcuding some reads longer than 100 kbp, were sequenced, indicating the excellent integrity and quality of the high molecular weight DNA extracted with the kit.
High quality with market-leading results DNA was purified from HeLa cells by enzymatic lysis and analyzed on a Single Molecule Real Time (SMRT) sequencing platform (PacBio RS II). The read length of NucleoBond® HMW DNA (MN) was compared with two commonly used competitor products (kit P and kit M both from competitor Q). The use of NucleoBond®technology results in superior mean subread lengths, enabling longer reads and better sequencing results.

Optimal results from various samples using NucleoBond® HMW DNA

In addition to molecular weight and integrity, purity and yield are critical for good results with HMW DNA sequencing and are often an issue with the common methods for HMW DNA extraction. Diverse samples were extracted using the NucleoBond® HMW DNA kit. All extractions yielded in sufficient amounts of DNA for sequencing experiments. Absorbance ratios were A260/A280 >1.75 and A260/A230 >2.00, indicating high purity of the samples.

 

Features

NucleoBond® HMW DNA
Technology	Anion exchange chromatography
Format	Midi gravity flow columns
Fragment size	Up to 200 kbp with > 90% depletion of fragments < 10 kpb
Sample material	< 1.5 g plant leaves (ground under liquid nitrogen) < 107 cultured cells (enzymatic lysis) < 300 mg solid tissue (cut into small pieces and lysed enzymatically with increased lysis time, ground under liquid nitrogen, or lysed by bead beating) < 30 mg yeast or bacteria (ground under liquid nitrogen) < 300 mg yeast or bacteria (lysed enzymatically or by bead beating) < 2 mL liquid sample (e.g., blood, body fluids or enzymatic reactions)
Typical yield	Depending on the sample amount and type
Elution volume	50–250 µL
Processing time	2h/12 preps (including 30 min lysis)

 

Applications*

High molecular weight DNA from mammalian tissues, blood samples, invertebrates, plants, bacteria, and yeast

* Kits to be used for research purposes only