New England Biolabs, T3050S, Monarch® HMW DNA Extraction Kit for Cells & Blood

Related Categories High Molecular Weight DNA Extraction,, Nucleic Acid Purification FAQ Q: How does excess residual protein in blood and tissue preps affect the purification process? A: If there is excess protein in the lysate prior to the addition of isopropanol for the precipitation step, the contaminating protein inhibits proper binding of the DNA to the beads. Excess protein in the lysate causes the precipitated DNA to float within the lysate and does not allow it to bind to the beads efficiently. Additionally, the glass beads will tend to get stuck in the bottom of the 2 ml tubes. It is therefore very important to remove as much of the hemoglobin-containing supernatant as possible from the erythrocyte pellet during a blood prep; do not leave more than 20 μl behind. The same is true during tissue preps; exceeding the maximum input amounts will also lead to excess protein in the lysate and DNA will not bind efficiently to the beads. 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 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: Are the Monarch HMW DNA Extraction kits compatible with samples stored in Monarch DNA/RNA Protection Reagent, RNAlater, DNA/RNA Shield etc.? A: Yes. For RNAlater®, it is sufficient to remove the stabilization solution completely before processing. Subsequently, standard protocols can be followed. If working with Monarch DNA/RNA Protection Reagent (NEB #T2011) or Zymo’s DNA/RNA Shield®, the reagent should be used as 1X dilution for lysis, rather than the lysis buffer supplied in the kit. In general, however, results obtained with fresh and frozen materials are superior over stabilized samples in terms of yield, purity and fragment length. 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 I still use the Monarch HMW DNA Extraction Kits if I do not have a thermal mixer? A: If the goal is to obtain DNA of maximal size, shaking during lysis in the thermal mixer is not required. However, Proteinase K digestion needs to be carried out at 56°C. To ensure proper lysis, invert your sample a number of times slowly during the proteinase K incubation. Please note that samples lysed without agitation will be very viscous and difficult to handle. If the goal is DNA tuned to 50 kb-250 kb (optimal for ligation-based nanopore sequencing), then we strongly recommend working with a thermal mixer (e.g., Eppendorf® ThermoMixer® C). This will result in a controlled shearing to the optimal fragment size range. If access to a thermal mixer is not possible, the best alternative for agitation in the thermal mixer is to pulse-vortex for one minute at the highest speed possible after the lysis incubation. For tissue samples, briefly vortex the sample every minute or so during the beginning of the lysis incubation, until there are no tissue pieces remaining. Then, vortex again for one minute after the lysis incubation. 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 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 mouthwash samples? A: We have not tested this, but we would expect it to work with the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050). The cell population in a mouthwash sample would typically consist of buccal epithelial cells and white blood cells. These can be pelleted as recommended in the cell protocol and we would recommend washing the pellet with cold PBS 1 or 2 times. It will be important that there are enough cells present, so it may require a large volume of starting material, as the cell density (and hence the DNA amount) in mouthwash tends to be low. 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: Do you have any PacBio Sequencing results from DNA extracted from tissue samples with the Monarch HMW DNA Extraction Kits? A: Yes, we have carried out comparative experiments with PacBio SMRTbell libraries prepared with DNA from cells (HEK293), blood (porcine), bacteria (E. coli) and tissue samples (mouse kidney). In barcoded runs, all sequencing metrics were very similar. Libraries prepped with Monarch-purified HMW DNA have been run with excellent metrics on the Sequel I and the Sequel II. The DNA performed well in traditional and HiFi sequencing runs. 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 you use the DNA isolated with the Monarch HMW DNA Extraction Kits in Southern blots? A: Yes, the extracted DNA can be used for Southern blot analysis. Make sure to separate the HMW DNA on a pulsed-field gel, as on regular agarose gels the HMW DNA will get stuck in the gel slots. 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: My blood sample is frozen in aliquots larger than is recommended in your protocol (e.g., in Vacutainers). Can I still use the frozen blood protocol for the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050)? A: Yes, but it is important not to thaw the sample completely (or warm up) when it is not in the presence of RBC Lysis Buffer, as allowing the sample to warm will quickly activate nucleases. Incubate the blood container at room temperature until it is just about to start thawing (the color will become darker red). Place the tube in a tube rotator set at 20 rpm and rotate until the sample has almost thawed completely. Be sure to monitor the sample on the rotator very carefully to catch the point at which it is almost thawed. At that point, vortex briefly to finalize thawing and dissolve any aggregates and quickly place on ice. Take an aliquot of blood for your prep (recommend starting volume is 500 µl) and place in a 2 ml tube. Add 3 volumes of cold RBC Lysis Buffer, mix by inversion 5 times, and then continue with the frozen blood protocol (Step 5). Thawed blood samples should not be re-frozen. Alternatively, you can thaw the blood sample ever so slightly in a 37°C water bath, just enough to dislodge it from the container and move it into a larger container capable of accommodating the addition of 3 volumes of cold RBC Lysis Buffer. Then, the sample can be thawed in the presence of RBC Lysis Buffer according to the protocol. Please note that this approach may require additional RBC Lysis Buffer (NEB #T3051L). 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. Q: The enzymes in the kit were not stored at -20°C right away. Will they still work? A: Yes, unopened Monarch Proteinase K and RNAse A enzymes are stable at ambient temperatures short-term. After opening, Proteinase K and Monarch RNAse A should be stored at -20°C. Q: There is a precipitate in the Proteinase K enzyme tube, is this normal? A: It is not uncommon to see a white, amorphous precipitate in the Proteinase K tube. There is no impact to Proteinase K activity or kit function, so you can proceed as normal with your workflow. Q: Which Monarch® columns and bead retainers can be used with which Monarch collection tube? A: Monarch Spin Collection Tubes (NEB# T2118) are compatible with Monarch RNA Purification Columns (NEB #T2007); Monarch gDNA Removal Columns (NEB #T2017); Monarch RNA Cleanup Columns (NEB #T2037, #T2047, #T2057); Monarch gDNA Purification Columns (NEB #T3017); and Monarch Bead Retainers (NEB #T3004). Monarch Collection Tubes (NEB #T1018) are compatible with Monarch Plasmid Miniprep Columns (NEB #T1017), and Monarch DNA Cleanup Columns (NEB #T1034). Monarch Collection Tubes II (NEB #T2018) are compatible with Monarch RNA Cleanup Columns (NEB #T2037, #T2047, #T2057) (through specific lots); Monarch gDNA Purification Columns (NEB #T3017) (through specific lots); and Monarch Bead Retainers (NEB #T3004) (through specific lots). Q: I noticed the collection tube dimensions has changed slightly in the Monarch® HMW DNA Extraction Kit for Cells & Blood (NEB #T3050) and the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060). Will this impact performance? A: NEB is standardizing the number of plastics we use in our kits to decrease waste during their production. The slight changes to dimension will not impact product performance. Q: I notice that I'm applying force to fit the column into a collection tube. Should I be doing this? What collection tubes are compatible with columns? A: Matching collection tubes are provided in all Monarch® kits for the spin columns. If you use collection tubes outside of a given kit, please use the Monarch® Spin Collection Tubes (NEB #T2118). Do not force any columns into non-recommended collection tubes that have a very tight fit to avoid damaging the column. Q: I have used all the Proteinase K (NEB #P8200) included in the Monarch kit. Can I purchase more? A: Yes, Proteinase K, Molecular Biology Grade (NEB #P8107), is available for standalone purchase and can be used as a direct substitute for (NEB #P8200) in all Monarch protocols without any modifications.