Measuring and Analyzing HMW DNAHigh molecular weight genomic DNA is often viscous and challenging to handle and transfer volumes with accuracy. Before measurement, samples should be properly homogenized following the guidelines in Homogenization of High Molecular Weight DNA (HMW DNA) Samples. Homogenization prior to quantitation is particularly important if samples have been frozen for long term storage, where gDNA is unevenly distributed upon thawing. When measuring thawed samples, allow them to reach room temperature and homogenize to enable consistent measurements.
Samples prepared at high agitation speed are suitable for concentration and purity assessment on microvolume spectrophotometers (MVS) (e.g., Nanodrop®) after they have been homogenized. These samples should be briefly vortexed to ensure even distribution of the gDNA in the solution before quantitation; a short vortex will not shear DNA.
Samples obtained with low agitation speeds (UHMW DNA) tend to be less homogeneous and may be more challenging to handle and apply on MVS systems. For these samples, mix with a wide bore pipette tip to ensure an even distribution of DNA within the sample before quantitation.
UHMW DNA needs to be measured several times, as complete homogenization is challenging. It is recommended to repeat the measurement ~5 times and use the average to assess the sample concentration. Chip-based systems like Unchained Labs® Lunatic (formerly known as Trinean® Dropsense® 16) are not suitable, as the viscosity of the samples prevents proper movement in the channels of the chip. In such cases, shear a part of the sample to reduce viscosity (see Homogenization of Homogenization of High Molecular Weight DNA (HMW DNA) Samples). Alternatively, a fluorescence-based quantitation system (e.g., Qubit®) can be used.
Spectrophotometric analysis of gDNA eluates can be used for assessing the quantity of the isolated gDNA by measuring the absorbance at 260 nm. As the Monarch HMW DNA Extraction Kit protocol efficiently removes RNA, 260 nm absorbance values provide an accurate indication of the amount of DNA present. Typically, modern micro volume spectrophotometers (e.g., Nanodrop) automatically calculate the DNA concentration by multiplying the measured absorbance value with the conversion factor, which is 50 for DNA. Concentration measurements at 260 nm can be performed on most microvolume systems down to 1 ng/µl with acceptable accuracy. Below that concentration, the use of fluorescence measurement via Qubit or similar detection systems is recommended. Please note that the A260/230 and A260/280 ratios are typically not reliable below 20 ng/µl. If DNA is not completely dissolved, it may be detected in the OD measurement as turbidity, which will result in reduced A260/230 values that may be falsely interpreted as impurity.
Analysis by standard gel electrophoresis or other electrophoretic methods (Labchip®, Bioanalyzer®, Tape Station®) may not provide suitable resolution to accurately assess the distribution of fragment sizes in the eluted sample. Typically, more than 80% of the material is ≥ 50 kb in length. Resolution of high molecular weight gDNA is best performed by pulsed-field gel electrophoresis on agarose gels. Faster methods that have some utility include pulsed-field capillary methods (e.g., FemtoPulse®).
Storage of HMW DNAThe Monarch gDNA Elution Buffer II (NEB #T3056) provided with the kit (10 mM Tris-HCl, pH 9.0, 0.5 mM EDTA) was developed as a long-term storage buffer. The combination of EDTA and high pH provides optimal protection against nucleases. If the sample will be actively used, it is recommended to store HMW samples at 4°C; for long term storage, store at -20°C. Avoid repeated freeze thawing and always use low bind tubes to prevent DNA from binding to the tube walls.
- Monarch HMW DNA Extraction Kit for Cells & Blood
- Monarch HMW DNA Extraction Kit for Tissue
- Monarch HMW gDNA Elution Buffer II
- Monarch HMW DNA Extraction Kit for Cells & Blood Product Manual
- Monarch HMW DNA Extraction Kit for Tissue Product Manual
- Homogenization of High Molecular Weight DNA (HMW DNA) Samples
- Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
- Troubleshooting Guide for High Molecular Weight DNA Extraction Using the Monarch HMW DNA Extraction Kits
- Guidance for HMW DNA Extraction Upstream of Oxford Nanopore Technologies®’ Ultra-Long DNA Sequencing Workflow