New England Biolabs, T3056L, Monarch® gDNA Elution Buffer II

Related Categories High Molecular Weight DNA Extraction,, Genomic DNA Extraction & Purification,, Nucleic Acid Purification FAQ Q: Which measurement system is best used for assessing the concentration of HMW DNA? A: In our experience, and in contrast to common belief, measuring the concentration of HMW DNA by Qubit® is not the most accurate approach. OD measurements provide more accurate concentration values, but because of the viscosity of HMW DNA samples, several replicates are required. Qubit readings will typically underestimate the amount of HMW DNA; for genomic DNA isolated with silica spin columns (usually 20-80 kb), Qubit provides DNA concentration readings 10-15% lower than those obtained by spectrophotometer. The ability of the intercalating dye to stain the DNA decreases with increasing DNA fragment length, resulting in an even larger difference between Qubit and OD values when working with HMW DNA. Differences can be as large as 35%, or even higher in some cases, resulting in a significant underestimation of DNA concentration. Q: I am having trouble dissolving my HMW DNA after isolation. Do you have any tips on increasing the solubility? A: High molecular weight DNA (HMW DNA) is notoriously difficult to dissolve. When DNA samples are highly concentrated or contain ultra-high molecular weight DNA, dissolving it can be even more challenging. In general, the more DNA is compacted, the harder it is to get into solution. The glass beads employed in the Monarch HMW DNA Extraction Kits provide a large surface for the DNA to wrap around which generally serves to keep the DNA in an open conformation, making it easier to dissolve. However, solubility can still be an issue. Below are some tips to maximize the solubility of your DNA when working with the Monarch HMW DNA Extraction Kits: Try to minimize the amount of time that the DNA is bound to the beads without liquid being present. After removing liquid from the beads, add the next buffer without delay. The longer the DNA is dried to the beads, the more compact the DNA will be and the more difficult it will be to dissolve later. When binding the DNA to the beads, do not invert the sample longer than recommended. The more time that the DNA wraps around the beads, the more compacted it will get, and the more difficult it will be to dissolve later. Review our usage guideline, Homogenization of High Molecular Weight DNA (HMW DNA) Samples After Elution for more tips on homogenization of HMW DNA after elution.