HMW DNA can be isolated from fresh or frozen tissue. In general, fresh tissue samples should be processed immediately. If processing of fresh tissue samples is delayed for several hours, the quality of the isolated HMW DNA may be lower, particularly for metabolically active organ tissues. Alternatively, samples may be frozen and stored at -80°C. If working with fresh or frozen tissue pieces, ensure the tissue is cut into the smallest pieces possible.
Adequate sample storage can be carried out by one of two methods: A) Flash frozen tissue samples can be stored as whole pieces at -80°C. B) Flash frozen tissue samples can be pulverized in liquid nitrogen using a mortar and pestle, and subsequently stored at -80°C as tissue powder. Frozen tissue powders are recommended over frozen tissue pieces, as lysis is more efficient, and handling is easier. Freshly or recently prepared frozen tissue powder samples provide better yields than those stored long term at -80°C. Working with stabilized tissue (e.g., RNAlater®) is possible, but is not the preferred option, as fragment sizes may be reduced when compared to fresh and frozen tissue samples.
Fresh Tissue Samples
- Weigh out the exact amount by transferring small tissue pieces into a reaction tube positioned on a micro balance.
- Keep tubes cold and process as soon as possible.
- Do not use more tissue than recommended (see “Choosing Input Amounts for the Monarch HMW DNA Extraction Kits”).
Frozen Tissue Powders
- Label and pre-cool reaction tubes on dry ice. Keep tubes containing tissue powder on dry-ice and use small pre-chilled scoops that allow for the transfer of 2 or 10 mg frozen tissue powder at a time.
- Tare pre-chilled tube on the micro balance and transfer the appropriate amount of frozen tissue powder to tube for weighing. Do not use more tissue than recommended (see “Choosing Input Amounts for the Monarch HMW DNA Extraction Kits"). Work quickly to prevent the tube from warming up on the balance.
- Keep the aliquoted samples on dry ice to ensure the powder stays frozen.
Frozen Tissue Pieces
- Keep samples frozen (e.g., by storing on dry ice).
- Use a clean, frozen cooling block or the bottom side of a frozen metal microfuge tube stand for cutting frozen tissue. Samples are most easily cut when they are processed shortly before thawing.
- Weigh the desired amount by transferring small tissue pieces into a pre-chilled reaction tube positioned on a micro balance. Do not use more tissue than recommended (see “Choosing Input Amounts for the Monarch HMW DNA Extraction Kits”)
- Keep tubes frozen or on ice, and process as soon as possible. In samples that have been frozen, ice crystals have destroyed cell structures and nucleases have free access to the genomic DNA. Work with the smallest possible tissue pieces to allow for efficient homogenization and rapid inactivation of nucleases by Proteinase K.
Stabilized Tissue Pieces
Tissue samples that are stored in stabilization reagents (e.g., Monarch DNA/RNA Protection Reagent (NEB #T2011), DNA/RNA Shield, RNAlater) can also be processed. However, working with fresh or frozen tissue without stabilization reagents is recommended for optimal purity, yield, and longer, more uniform DNA fragment size.
When working with samples stored in RNAlater, completely remove the reagent from the tissue sample and follow the standard protocol. If working with samples stored in 2X Monarch DNA/RNA Protection Reagent (NEB #T2011) or in 2X DNA/RNA Shield, remove the reagent from the tissue sample, transfer it to a different tube, and dilute it to 1X with nuclease-free water. Homogenize the tissue sample with the supplied pestle and tube. Add Proteinase K to the 1X reagent and use this in place of the Monarch HMW gDNA Tissue Lysis Buffer supplied with the kit. Unfortunately, sample lysis will be incomplete and small tissue pieces may still be visible on completion of the lysis incubation. Adding 1/10 volume of Monarch Proteinase K Reaction Buffer, which is a component of the Monarch Total RNA Enzyme Pack (NEB #T2019L), to the 1X solution will increase lysis efficiency. Upon adding the Protein Separation Solution, the subsequent centrifugation step will not result in phase separation; instead, a pellet with small tissue particles will form. Transfer all of the supernatant to a new 2 ml tube and follow the standard protocol.
Special Considerations for Specific Tissue Types
Liver samples are rich in the polysaccharide, glycogen. When isolating DNA from liver samples, glycogen may coprecipitate with DNA and affect DNA purity, resulting in variable or low A260/A230 values. Addition of the appropriate concentration of salt (NaCl) to a liver lysate before DNA precipitation helps to remove polysaccharides. Specifically, when DNA is precipitated in the presence of 2.2 M NaCl, polysaccharides remain in solution and are discarded, typically resulting in purified DNA with A260/A230 values > 2.0. This protocol modification is provided after Step 11, in Part 1 of the Tissue Protocol.
Brain is a fatty tissue. During pestle homogenization, the fatty material tends to stick and may accumulate on the upper rim of the pestle. For maximal yields, this tissue material should be returned to the lysate using the tip of a p200 pipette tip. Reducing agitation time from 45 minutes to 15 minutes during lysis may improve yields from brain samples; yields may be up to 50–100% higher, particularly when working with input amounts close to the lower end of the input range. The high fat content of brain samples also affects the phase separation and typically requires longer centrifugation times to be complete. Chilling the lysed sample for 3 minutes before adding the Protein Separation Solution will facilitate the phase separation and isolated DNA will be of higher purity. The high fat content of brain samples limits the maximum input amount to 20 mg; above that, a complete phase separation will not be achieved with the given liquid volumes.
Muscle is a fibrous tissue. Reducing agitation time from 45 minutes to 15 minutes during lysis will improve yields from muscle samples; yields may be up to 50–100% higher, particularly when working with input amounts close to the lower end of the input range. During lysis, insoluble protein fibers will float on the surface of the upper phase after the phase separation. For highest purity, avoid transferring these fibers. A simple way to get rid of a substantial amount of the fibers is to dip the p1000 wide bore tip into the upper phase when starting the transfer of the upper phase. Many fibers will stick to the outside of the pipette tip and will remain stuck to the plastic material during the further transfer process.
Mouse and Rat Tail Tips and Ear Punches
Mouse and rat tail tissues are very rigid and fibrous, making homogenization and lysis challenging. As rotor-stator homogenization is not effective for this tissue type, manual homogenization with a microtube pestle is recommended. However, even intensive grinding with a pestle will not allow for complete homogenization because of the toughness of the tail material. Before starting homogenization and lysis, tail samples should be cut into very small pieces (1-2mm) with surgical scissors or a razor blade. Lysis at the highest agitation speed (2000 rpm) is recommended to speed up lysis. However, for isolation of the largest possible HMW DNA, agitation can be stopped or reduced to 500 rpm once the tissue pieces are fully lysed (5-10 minutes). Because of the challenges with lysis, HMW DNA isolated from tail tips tends to have a lower average length than DNA from organ tissue. Depending on the downstream application, a size selection step may be considered.
20 mg is the recommended input amount for tail tips, although up to 25 mg (~8 mm tail tip) can be processed; expected yields are ~ 2 μg per mg. Tail tissue becomes more rigid with increasing animal age, and as such, best results are obtained with younger animals. Fresh samples will yield higher DNA quality than frozen ones, as tissues that have been previously frozen are more susceptible to nucleases during lysis.
Like tail tips, ear punches are also tough and difficult to lyse. Ear punches should be cut into very small pieces to facilitate lysis, which, in contrast to organ tissue, will take several minutes. During lysis, ear punches (and skin tissue in general) will release insoluble fibers which tend to float on the surface of the upper phase after the protein removal step; avoid transferring any of these fibers. The recommended input amount is 10 mg, and because of the fiber issue, not more than 15 mg should be used. Samples < 10 mg should be processed as “low input” samples with reduced lysis volumes. Expected DNA yield is ~1.5 µg per mg tissue.
In addition to tissue, the Monarch HMW DNA Extraction Kit for Tissue provides a rapid and reliable process for extracting HMW genomic DNA from bacteria. Separate protocols for processing Gram-negative and Gram-positive bacteria are provided and differ slightly in the initial lysis step. Lysozyme is required to efficiently lyse the bacterial cell wall in these tough-to-lyse samples, and for processing Gram-positive bacteria, a STET buffer is also required. Alternative lysis enzymes (e.g., lysostaphin) may be required for certain Gram-positive bacteria. Ensure that the recommended input amounts of cells are used by measuring the density of bacteria cells in liquid culture (see “Choosing Input Amounts for the Monarch HMW DNA Extraction Kits”). Cells should be pelleted, and all culture medium removed prior to sample processing. Standard and low input protocols are provided to ensure the buffer volumes are appropriate for the sample input amount used.
- Monarch HMW DNA Extraction Kit for Tissue Product Manual
- Choosing Input Amounts for the Monarch HMW DNA Extraction Kits
- Choosing an Agitation Speed During Lysis with the Monarch HMW DNA Extraction Kits
- Homogenization of High Molecular Weight DNA (HMW DNA) Samples
- Measuring, Analyzing & Storing High Molecular Weight DNA (HMW DNA) Samples
- Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
- Considerations for Loading Pulsed-Field Gels (PFG) with High Molecular Weight DNA)
- Troubleshooting Guide for High Molecular Weight DNA Extraction Using the Monarch HMW DNA Extraction Kits