New England Biolabs, T3005L, Monarch® DNA Capture Beads

Related Categories High Molecular Weight DNA Extraction,, Genomic DNA Extraction & Purification,, Nucleic Acid Purification FAQ Q: I dropped a few of my glass beads. Can I clean them, and do you supply extra? A: NEB provides an overage of beads to accommodate for users who may drop them. In the 5-prep kits, we provide 2 extra beads, and in the 50-prep kits, we supply ~5-7 extra beads. The Monarch DNA Capture Beads are also available for purchase separately (NEB #T3005). Additionally, any beads that are dropped can be washed with detergent solution (e.g. 2% SDS or RNaseZap), rinsed water and with 70% ethanol, dried and then used. Q: Is there anything special about the Monarch DNA Capture Beads, or can I use any glass beads? A: The Monarch DNA Capture beads are specialized borosilicate glass beads optimized for extraction of HMW DNA in the Monarch extraction workflow. Even though other glass beads may successfully purify HMW DNA, the Monarch Capture Beads provide optimal performance in downstream applications like Nanopore sequencing. We cannot guarantee equivalent performance using any other glass beads besides those provided. Q: How do I know that my HMW DNA has attached to glass beads in the Monarch HMW DNA Extraction Kits? A: When samples contain >10 µg of HMW DNA, which is common when working with ‘Standard Input’ amounts, the binding of the DNA to the beads is visible and will be seen as a cloudy, opaque layer covering the beads. The beads will always stick together after this amount of DNA is bound to them. When inversions are done manually using ‘Standard Input’ amounts, the binding process can be seen and followed. After 5 inversions, the cloud-like DNA becomes visible, and in the next 10 inversions, it connects to the beads and starts wrapping around them. At this point, the solution remains viscous. Once the DNA is completely wrapped around the beads during the remaining 10-15 inversions, the solution will no longer be viscous. ‘Low Input’ samples typically yield between 1-10 µg of HMW DNA and the binding of the DNA may be more difficult to follow. If only 1-5 µg of DNA is present, it will likely not be possible to see the DNA binding to the beads; the beads will seem to move freely and independently in solution. When there is between 5-10 µg of DNA, it is difficult to see the DNA binding to the beads, but the bound DNA will often cause the beads to stick together. Q: Is there any benefit to adding in a size selection (e.g., Circulomics® Short Read Eliminator) following extraction? A: In NEB’s experience, a size selection step is usually not necessary upstream of ligation-based nanopore sequencing. When working with fresh, high-quality starting materials, the N50 read lengths are high (35 – 55 kb, depending on the sample) and an additional size selection of the HMW DNA will provide only marginal improvement in read length. However, when working with starting materials of lower quality, for example those which have been stored sub-optimally, or when working with samples that are very difficult to lyse (e.g., mouse tail) the resulting DNA will contain a larger portion of short fragments. In these cases, it may be useful to carry out an enrichment step for better sequencing results; any size selection product (e.g., Short Read Eliminator) can be used with the DNA isolated with the Monarch HMW DNA Extraction Kits. Q: Can the Monarch HMW DNA Extraction Kit be used for processing yeast and/or fungal samples? A: We have validated our kit for processing yeast samples and have published a protocol based on working with S. cerevisiae samples. We have not internally tested the kit on filamentous fungi. However, based on our work in developing our silica kit (NEB #T3010), which uses similar lysis chemistry, we found our chemistry may work for certain fungi when the sample is frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. For mushrooms, dried starting material should be used. However, this approach would need to be tested for your specific species. Q: Can the Monarch HMW DNA Extraction Kits be used to process human Buffy coat? A: Yes, the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050) can be used for processing Buffy coat. When working with Buffy coat, which is a concentrated PBMC solution, smaller input amounts should be used than when working with blood. The total cell count should not be higher than 1 x 107 cells for the standard protocol (agitation at 2000 rpm) and not higher than 5 x 106 cells when UHMW DNA is needed (low agitation). Buffy coat as obtained from blood banks contains up to 1 x 109 PBMCs per 30-80 ml, which equates to approximately 1 x 107 -3 x 107 cells/ml. We suggest starting with 100-300 μl of this solution, add PBS to a total volume of 500 μl, and move into the erythrocyte lysis procedure as if it were a 500 μl blood sample. Q: Can the Monarch HMW DNA Extraction Kits be used to process environmental or fecal samples for metagenomic studies? A: We do not recommend our kit for processing environmental samples like soil or for processing fecal samples. These samples have two main challenges: they both have a very high nuclease burden, and they contain inhibitors that are very difficult to remove (humic acid for soil, bile acids for fecal samples). The nuclease burden results in significantly degraded DNA, which is inherently not suitable for HMW DNA isolation, and the inhibitors present in those samples will not be effectively removed by the current kit chemistry. Q: Can the Monarch HMW DNA Extraction Kits be used to isolate BAC DNA? A: We have not tested this, but expect this should work as the BAC constructs are in the optimal size range for bead binding. However, BACs usually are very low copy number constructs, and a significant amount of culture will be needed to accumulate enough BAC DNA for the bead binding (> 5 μg). This will lead to an increase in the buffer volumes needed in the alkaline lysis. Altogether, the volume of the 3 alkaline lysis buffers should not surpass 1100 μl so that the DNA can still be bound to the beads in a 2 ml reaction tube. Some optimization work may be required, but it should be possible to connect the alkaline lysis chemistry with the bead binding and purification part of the Monarch protocols. Q: Does the Monarch HMW DNA Extraction workflow work for single cells? A: Unfortunately, this workflow is not compatible for use with single cells. The system requires the presence of a critical mass of DNA (≥ 1 μg in ~100 μl) for effective binding to the beads. Q: Can the Monarch HMW DNA Extraction Kits be used to process nematodes? A: Yes. We have successfully isolated HMW DNA from C. elegans with the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060) using the standard protocol for tissue samples (worms from 2 plates were processed). We recommend using a rotor-stator homogenizer for sample homogenization for best yields. 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.