New England Biolabs, T3061L, Monarch® HMW gDNA Tissue Lysis Buffer

Related Categories High Molecular Weight DNA Extraction,, Nucleic Acid Purification FAQ Q: Can I use the Monarch HMW DNA Extraction Kits for processing plant samples? A: We do not recommend this kit for DNA extraction from plants. Polysaccharides in the plant lysates are not effectively removed by the included HMW gDNA Tissue Lysis Buffer, the lysis buffer included in the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060). The presence of residual polysaccharides prevents the binding of the DNA to the glass beads. If you have your own lysis system that could be employed in conjunction with our workflow, we certainly encourage you to test this. Our initial work highlighted the importance of starting with at least 50 mg of plant tissue because the binding efficiency to the glass beads requires a critical mass of DNA. Using smaller input amounts will not provide sufficient DNA to allow binding to the beads. If a nuclei preparation is made from plant samples, please choose the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050) to process the plant nuclei (NEB has not validated this approach but welcome users to try it). If the plant nuclei are present in a dedicated buffer, use this buffer instead of the Nuclei Prep Buffer. Alternatively, pellet nuclei and resuspended in Nuclei Prep Buffer and follow the protocol from there. We encourage you to share your feedback on testing our technology with plant samples by emailing us at info@neb.com. Q: Can I use the Monarch HMW DNA Extraction Kits for processing insect samples? A: We have successfully isolated HMW DNA from A. aegypti (15 mg) using the standard tissue protocol. Though we have not internally validated any other insects at this time, others have successfully processed flies, moths, arthropods (scorpion) and some nematodes. The following considerations may be helpful: Work with fresh samples if possible; freeze thawing can activate nuclease activity When working with larger insects, we recommend removing the gut material (which is rich in nucleases) before homogenization Thorough homogenization with pestle or rotor stator homogenizer is essential, and quickly adding the lysis buffer (which removes nucleases) after homogenization is important 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 marine samples, including marine invertebrates? A: We have not tested the kit on these organisms. Often these organisms contain a lot of mucus/ polysaccharide-based material, which may cause problems during bead binding. If you have an optimized lysis chemistry, you may be able to employ that upstream of our bead workflow using the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060). 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: How do you suggest homogenizing tougher tissues, like ear punches or muscle? Can a sonicator be used for tissue homogenization? A: For sample homogenization, we recommend the included microtube pestle or rotor-stator homogenization. Sonication is not recommended for the extraction of HMW DNA as it is known to shear DNA. For homogenization of fibrous tissues, we have the following recommendations: Muscle can be homogenized with either the microtube pestle or a rotor-stator homogenizer. We consider working with the pestle more convenient, but for frozen samples, the rotor-stator homogenizer gives somewhat better N50 read lengths. Ear punches and tail tips work better with the microtube pestle, as the rotor-stator homogenizer with the disposable tips (Tissueruptor II) is not able to effectively disrupt the tissue. It is possible that the rotor-stator homogenizer with the non-disposable sharp metal tips could be a better option, but we have not tested that. However, we have obtained excellent results with ear punches using the microtube pestle. Essentially, the pestle splits the ear punch sample in three thin layers that are accessible to the tissue lysis buffer and Proteinase K, providing very good quality HMW DNA. We recommend using 2 ear punches per sample for highest binding efficiency. Tail tips are significantly challenging samples. Ideally, use a powder made by grinding with liquid nitrogen. But if that option is not available, the pestle does help, but it still takes the Proteinase K several minutes to lyse the homogenized tissue completely. As a result, the DNA isolated from mouse tail is more sheared and smaller in size than that isolated from ear punches. Accordingly, a short read eliminator step may lead to better read lengths in nanopore sequencing. Q: Does the kit work with frozen tissue, tissue stored in preservatives (ethanol, DMSO-salt/DESS, or RNAlater®), FFPE samples, or OCT-preserved tissues? A: We have successfully tested several of the options for preservation of frozen tissue samples, but fresh input material is always best whenever possible. When fresh samples are not available, freezing is the recommended alternative, ideally shock freezing with liquid nitrogen and storing at -80°C. When thawing, the goal is not to give the nucleases much time to harm the DNA, and you can ensure this by carrying out the homogenization rapidly and mixing with the lysis buffer immediately after the sample is thawed and homogenized. Interestingly, frozen tissue samples often give better results in terms of DNA fragment length and integrity when compared to those stored in preservation reagents. We have tested Monarch DNA/RNA Protection Reagent (NEB #T2011), RNAlater, DNA/RNA Shield, and RNAssist. Although these reagents effectively preserve the samples, their presence prevents rapid digestion of the tissue material during the initial phase of lysis, which seems to result in some shearing/DNA damage. In our experience, the Monarch DNA/RNA Protection Reagent provides somewhat better results than RNAlater and RNAssist. Ethanol-preserved tissue can be used as input material. Before starting the lysis procedure, ethanol should be removed by washing samples with a medium-salt buffer; further details are available on request (contact us at info@neb.com). DMSO-preserved frozen cells have been tested and gave good quality DNA. We have not tested DESS but presume that it should work as well. FFPE samples are not suitable for HMW DNA isolation as they are highly crosslinked, resulting in significant fracturing and denaturing of the DNA during the stringent purification workflow that is required for these samples. Typically, FFPE samples are not a target input material for those seeking HMW DNA, since purified DNA will mostly be < 1 kb and will be denatured. OCT samples may potentially work, as the OCT fixed samples are not crosslinked, but we have not tested this. We encourage users to give it a try using our free trial kits. It is always good to stay in the middle range of the recommended input amount (e.g., 10-15 mg) to start. Before starting the DNA extraction, remove as much OCT as possible. Q: Do you see different fragmentation results when using the pestle versus the rotor-stator homogenizer for tissue homogenization? A: If the goal is to isolate ultra HMW DNA, microtube pestle homogenization is the best choice. Rotor-stator homogenizer (RSH) is an efficient alternative, but the HMW DNA that is released into the lysate may undergo a mild degree of shearing during the homogenization process. Therefore, for recovering the longest DNA fragments using the RSH, it should be used at the lowest setting and the device should be switched off immediately once the tissue pieces are completely disrupted. Generally, for pestle homogenization, the more thoroughly a sample is homogenized, the shorter the lysis time will be and the larger, more intact the DNA will be. The sample should be ground into the thinnest possible layer so that lysis by Proteinase K happens quickly. However, the tissue structure also influences the speed of lysis, and as a consequence, the fragment length. Soft organ tissue and brain will lyse in seconds and will give the longest fragments; muscle tissue may need a bit longer but should be lysed within a minute or so. Very rigid fibrous tissue (e.g., mouse tail) needs several minutes to lyse completely and therefore tends to give shorter DNA fragments. Since tissue cells cannot be freely resuspended as cultured cells can, portions of the cells lyse simultaneously, causing a degree of tangling of the HMW DNA. This tangling can be observed as upward smearing in a pulsed-field gel lane. Rotor-stator homogenizer treatment can cut those aggregates apart, for the most part. Pestle-treated samples however, particularly when frozen tissue is used, may require some needle shearing to resolve these DNA aggregates in order to get optimal long read sequencing results. Pestle and rotor-stator homogenization techniques produce different fragment lengths HMW genomic DNA was purified from soft organ tissues (mouse kidney, mouse liver, 10 mg), fatty (mouse brain, 20 mg), and fibrous (mouse muscle, 20 mg) tissues along with Gram – and Gram + bacteria (E. coli, 2 x 108 cells and B.cereus, 2 x 109 cells) were purified using the Monarch HMW DNA Extraction Kit for Tissue with various homogenization methods: pestle (P), rotor-stator homogenizer TissueRuptor II) (R), or thermal mixer (T). 300 ng DNA of each sample was resolved on a Pippin Pulse™ gel (Sage Science) with the program for 5–430 kb. Vigorous shearing induced by the rotor-stator homogenizer reduced fragment length compared to pestle homogenization + agitation in a thermal mixer.