Troubleshooting Guide for Monarch HMW DNA Extraction Kits (NEB #T3050 and #T3060)

Need some help purifying high molecular weight genomic DNA (HMW DNA) with the Monarch HMW DNA Extraction Kits (NEB #T3050 and #T3060)? We’re here to help. Our troubleshooting guide below outlines some of the issues that may be encountered during HMW DNA extraction. You can also find helpful guidance on choosing appropriate input amounts in our online resource, Choosing input Amounts for the Monarch HMW DNA Extraction Kits. Still having trouble? Contact our technical support at any time.

PROBLEM POSSIBLE CAUSES SOLUTION
LOW YIELD
  Input amount too low
  • Use recommended input amounts for best results. If using an input amount below the recommended amount, DNA recovery will be significantly less efficient
  Lysis volume too large
  • Use the appropriate lysis volume for the chosen input amount. The efficiency of binding to the glass beads is reduced when the DNA concentration is too low. Use of the appropriate lysis volume will establish optimal binding conditions and improve yields.
  DNA did not attach to the beads
  • In rare cases, the DNA may not attach to the beads and remains in solution during the binding step. Twist the tube sideways to create contact between the precipitated DNA and the beads. If DNA still does not attach to the beads, spin the precipitate down and carefully remove the supernatant. Add 500 µl of Monarch gDNA Wash Buffer, carefully invert the tube 2-3 times, spin down briefly, and then remove as much wash buffer as possible, without disturbing the DNA pellet. Pulse-spin in a minicentrifuge and remove residual wash buffer. Next, invert the tube and air dry at room temperature for 5 minutes. Add elution buffer and incubate for 5-15 minutes at 56°C in a thermal mixer with agitation at the lowest speed (300 rpm). Pipette up and down with a wide-bore pipette tip to homogenize. Leave overnight at room temperature for further dissolving or incubate for 30 minutes at 37°C and mix by pipetting again.
  Inaccurate quantitation of XL DNA concentration
  • Measuring XL DNA accurately is challenging, as the large DNA molecules may be unevenly dispersed in solution. When measured on a spectrophotometer, the samples often show a significantly lower concentration than what is truly present. Repeated measurements may give a more accurate concentration estimate. Please refer to “Measuring & Analyzing HMW DNA Samples” for more information. Alternatively, shearing a small portion of the sample to reduce the viscosity and homogenize the solution as described in “Homogenization of HMW DNA Samples”, to enable accurate quantitation.
Cells Input amount too low  
  • Working with < 1 x 105 cells is not recommended as recovery drops drastically below this number. Inputs in the range 1–5 x 105 cells require working with reduced buffer volumes for maximal recovery; follow “low input” protocol guidance.
Lysis volume too large
  • If working with ≤ 5 x 105 cells in the standard lysis volume, the efficiency of binding to the glass beads will be reduced as the DNA concentration is too low for efficient attachment. In such cases, a 3X reduction of the sample volume is needed. Use of the “low input” protocol will establish optimal binding conditions and improve yields.
Inaccurate cell count
  • Some types cultured cells (e.g., HEK293) tend to clump and may be difficult to accurately count. Clumping usually leads to an underestimation of the cell count and an unexpectedly high yield. If the cell count of dilute samples is incorrectly estimated to be above 5 x 105, the use of standard input protocols may lead to reduced efficiency of the prep and lower yield.
Cells and Blood Cells lost in pelleting step at supernatant removal
  • While spinning down the cells, be sure to note which side of the tube is pointing outside in the centrifuge, to identify where the cell pellet is located. The cell pellet may not be visible by eye when working with low cell numbers. Keep the cell pellet side of the tube facing downward and remove the supernatant from the upper side of the tube. Leave < 20 µl of supernatant behind so that the pellet is not disturbed.
Incomplete binding to the beads  
  • When working with high input samples (≥ 5 x 106 cells or ≥ 1 ml blood), especially when low agitation speeds are used for XL DNA, additional inversions are required for the DNA to bind completely and tightly enough to the beads. Increase the binding time in the rotator to 8 minutes. Insufficient binding can lead to loss of DNA during the following short spin step (to dry the beads).
Blood   Low input, small lysis volume needed
  • If working with < 500 µl blood (minimum is 100 µl) in the standard lysis volume, the efficiency of binding to the glass beads will be reduced as the DNA concentration is too low for efficient attachment. In such cases a 3X reduction of the sample volume is needed. Use the “low input” protocol to establish optimal binding conditions and improve yields. 
Leukocytes lost with supernatant removal during erythrocyte lysis
  • After spinning down the leukocytes during the erythrocyte procedure, be sure to note which side of the tube is pointing outside in the centrifuge, to identify where the leukocyte pellet is located. The pellet may not be clearly visible by eye when working with low input amounts or certain frozen blood samples. Keep the pellet side of the tube facing downward and remove supernatant from the upper side of the tube. Leave < 20 µl of supernatant behind so that the pellet is not disturbed.
Blood was thawed before adding RBC Lysis Buffer
  • Thawing blood samples before adding RBC Lysis Buffer will result in nuclease activity, leading to a significant reduction in DNA size and yield. Keep frozen blood samples frozen, add cold RBC Lysis Buffer directly to the frozen samples, and carefully follow the guidance in the protocol for handling frozen blood samples.
Blood sample is too old
  • Fresh (unfrozen) whole blood should not be older than a week. Older samples will show a progressive amount of DNA degradation and loss of yield.
Excess supernatant left behind after erythrocyte lysis
  • 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. The precipitated DNA will float within the lysate and cannot 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.
Tissue Incomplete homogenization
  • Thorough tissue homogenization is necessary for optimal sample lysis. When processing tissue samples, work with the smallest possible tissue pieces to allow for efficient homogenization and rapid inactivation of nucleases by Proteinase K. Use the included pestle or a rotor-stator homogenizer to thoroughly homogenize tissue samples prior to incubation in the thermal mixer.
Incomplete transfer of upper phase during protein separation
  • In the protein separation step of the protocol, HMW gDNA is present as a gradient in the upper phase with the majority of the DNA being concentrated in the fraction closest to the protein phase. Transfer as much of the upper phase as possible for best yields.
Shorter agitation time recommended for smaller input amounts
  • If using input amounts near the lower end of the input range, reduce agitation time from 45 minutes to 15 minutes during lysis. Finish the incubation time without agitation. This is particularly relevant for samples with low DNA content, like muscle and brain, and may increase yield by up to 50-100%.
Sample was not stored properly
  • 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 will be lower, particularly for metabolically active organ tissues.
  • Frozen samples should be kept frozen. In samples that have been frozen, ice crystals have destroyed cell structures and nucleases have free access to the genomic DNA. Snap freeze samples in liquid nitrogen to limit the damage to cell structures.
Sample not sufficiently homogenized
  • When processing fresh or frozen tissue samples, work with the smallest possible tissue pieces to allow for efficient homogenization and rapid inactivation of nucleases by Proteinase K. Ensure tissue material is homogenized into the thinnest layer possible when using the pestle. If using a rotor stator homogenizer, homogenize sample until all visible tissue pieces are gone.
DNA DEGRADATION
  Purified DNA sample kept at elevated temperatures too long
  • Although incubation at 56°C and 37°C helps to resuspend HMW DNA, particularly XL DNA, extended periods of heating HMW samples should be avoided, as this may result in DNA damage and eventually size reduction. Incubation time should not exceed 15–30 minutes for 56°C, 1–3 hours for 37°C and overnight incubation at room temperature. Samples are safe for long term storage at 4°C.
  Shearing introduced by inappropriate handling
  • XL DNA should always be pipetted using wide bore pipette tips and vortexing should be avoided. Extended heating at elevated temperatures will also negatively affect DNA fragment length negatively. DNA samples purified using maximum agitation speeds have shorter fragment lengths; these can be pipetted with regular pipette tips and can be vortexed briefly.
Blood Blood sample is too old
  • Fresh (unfrozen) whole blood should not be older than a week. Older samples will show a progressive amount of DNA degradation and loss of yield.
Blood sample was thawed before adding RBC Lysis Buffer
  • Thawing blood samples before adding RBC Lysis Buffer will result in nuclease activity, leading to a significant reduction in DNA size and yield. Keep frozen blood samples frozen, add cold RBC Lysis Buffer directly to the frozen samples, and carefully follow the guidance in the protocol for handling frozen blood samples.
Tissue Sample was not placed in thermal mixer immediately following homogenization and addition of lysis master mix
  • Sample homogenization initiates endogenous nuclease activity. After pestle or rotor-stator homogenization, samples in lysis master mix should be placed in the thermal mixer immediately. When working with multiple samples, each sample should be taken quickly through all homogenization sub-steps and placed in the thermal mixer before processing the next sample.
Sample was not stored properly
  • 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 will be lower, particularly for metabolically active organ tissues.
  • Frozen samples should be kept frozen. In samples that have been frozen, ice crystals have destroyed cell structures and nucleases have free access to the genomic DNA.
  • When processing fresh or frozen tissue samples, work with the smallest possible tissue pieces to allow for efficient homogenization and rapid inactivation of nucleases by Proteinase K.
  • Organ tissues like pancreas, intestine, kidney, and liver contain significant amounts of nucleases. They should be treated with extreme care and stored properly to prevent DNA degradation. Keep frozen and on ice during sample preparation.
ELUTED DNA DIFFICULT TO DISSOLVE
  Binding of DNA to the beads was carried out for too long
  • Do not exceed the recommended binding time (4 minutes in vertical rotating mixer or 8 minutes when working with high input amounts), as this will produce excessive compacting and tangling of the HMW DNA and lead to inefficient resuspension of the DNA during elution.
Cells and Blood Too much input material used
  • If the suggested input amounts are exceeded [1 x 107 for cells at max agitation speed, 5 x 106 cells for low agitation speeds, or 2 ml blood], the optimal relationship between bead surface area and efficient wrapping of the DNA around the beads is not maintained. This can produce excessive compacting and tangling of the HMW DNA and inefficient resuspension of the DNA during the elution step. Reduce the input amount per the recommendations. 
Incomplete resuspension or cell pellet at nuclei preparation step
  • Clumped cells that are not completely resuspended during the nuclei preparation steps may result in tangled DNA aggregates that cannot be dissolved completely after elution. Pay special attention to complete resuspension and lysis of the cells during the nuclei preparation step to obtain best results.
Blood Leukocyte pellets not completely resuspended
  • When working with frozen blood or blood that is several days old, leukocytes tend to be sticky and difficult to resuspend. Clumped cells that are not completely resuspended during the leukocyte pelleting steps and the nuclei preparation step may result in DNA aggregates that cannot be dissolved completely after elution. Ensure cells are completely resuspended after all pelleting steps to obtain best results.
Too much time taken for the erythrocyte lysis
  • Proceed through the erythrocyte lysis as rapidly as possible. Extended incubation with the RBC Lysis Buffer reduces leukocyte viability, causing stickiness and difficulty in resuspension.
PROTEIN CONTAMINATION
Blood Incomplete erythrocyte lysis
  • If the erythrocyte lysis steps are carried out too quickly, a substantial amount of hemoglobin will be carried over into the nuclei lysis (which is when the Proteinase K is added). Due to the larger amount of hemoglobin, the Proteinase K activity present may not be sufficient to degrade all of it, resulting in protein contamination. Follow the guidance on erythrocyte lysis times carefully to avoid protein contamination.
Too much supernatant left on leukocyte pellet
  • If too much supernatant is left on the leukocyte pellet during the erythrocyte lysis steps, a substantial amount of hemoglobin will be carried over into the nuclei lysis (which is when the Proteinase K is added). The amount of Proteinase K used in the nuclei lysis will be insufficient to handle that level of protein, resulting in protein contamination. Follow the detailed guidance on supernatant removal to avoid protein contamination.
Sample is very erythrocyte-rich (e.g., rabbit blood)
  • The blood of some animal species, like rabbit, is extremely rich in erythrocytes and these erythrocytes are not fully depleted during lysis. As a result, there will be significant carryover of protein into the nuclei lysis, and the capacity of the Proteinase K used during that step will be overwhelmed. Work with reduced input amount (200 µl instead of 500 µl) to ensure complete protein removal.
Tissue Protein carried over after phase separation
  • When transferring the phase containing the DNA (large, clear upper phase) to a new tube, do not transfer any material from the protein phase. It may be necessary to leave ~10 µl of the DNA phase in the tube to prevent carrying over the protein phase. If a lower protein phase is not visible, leave ~30 µl behind to ensure protein is not carried over.
Too much input material used, Proteinase K efficiency reduced
  • When sample input is exceeding the recommended amounts samples will be too viscous, resulting in reduced Proteinase K efficiency and incomplete protein removal.
Undissolved DNA falsely interpreted as protein contamination
  • If eluted DNA has not completely dissolved, this will result in some turbidity of the solution, that may be detectable by eye and will also affect the A260/A230 ratio of DNA samples. Upon complete dissolving of the DNA the A260/A230 will go back to higher values that are typically > 2.0 if the DNA is clean.
RNA CONTAMINATION
Tissue Too much input material used, RNase A efficiency reduced
  • When sample input exceeds the recommended amounts, samples will be too viscous, resulting in reduced RNase A activity. Do not use more than the recommended input amount.
RNase incubation step omitted in cell protocol
  • Cultured cells typically have high levels of RNA. To allow for complete RNA removal, the protocol includes a 2-step lysis procedure in which cell walls are lysed while nuclei are kept intact to keep the viscosity low in the lysate. This allows optimal performance of the RNase A to degrade the RNA that is released from the cytosol. Be sure to incubate samples with RNase A for at least 2 minutes at room temperature. Incubation is not necessary for leukocytes, as their RNA content is low following the erythrocyte lysis procedure.
NO PHASE SEPARATION
Tissue Low input amount used
  • With low input amounts, a protein phase may not be visible after centrifugation and phase separation. Use input amounts indicated in the protocol for best results. If a lower protein phase is not visible, leave ~30 µl behind to ensure that you do not carry over protein.
Protein is completely digested
  • In some cases, particularly when fresh tissue material is used, protein digestion may reach a high level of completion and no hydrophobic proteins will accumulate in a separate phase. Leave ~30 µl behind to ensure that you do not carry over protein.
INCOMPLETE PHASE SEPARATION
Tissue Too much starting material used
  • Follow the guidelines for tissue sample size. If the sample is not listed reduce input amount. Generally using more than 25 mg tissue is not recommended. For samples that are very DNA rich use no more than 15 mg.
Low agitation speed used
  • Additional centrifugation time (10-20 minutes) may be required for complete phase separation when low agitation speeds are used.
Fatty acid-rich samples need longer centrifugation time and prechilling
  • Pre-chilling lysates for 3 minutes on ice before adding Protein Separation Solution leads to more efficient phase separation. High input brain samples may require a longer centrifugation time for complete phase separation. Refer to “Special Considerations for Processing Specific Tissue Types: Brain”.
LOW A260/A230 0 (< 2.0) FOR LIVER SAMPLES
  Copurification of polysaccharides
  • Copurification of polysaccharides (i.e., glycogen) in liver preps results in variable or low A260/A230 values. Addition of NaCl to a liver sample before DNA capture helps to remove polysaccharides, resulting in A260/A230 values > 2.0. Refer to “Special Considerations for Processing Specific Tissue Types: Liver”.
Ratio A260/A230 > 2.5
  Slight variations in EDTA concentration in eluates
  • If the EDTA available in the elution buffer complexes with magnesium or calcium cations, which may be associated with the isolated genomic DNA in small amounts, this will lead to small differences in the free EDTA concentration in the eluate. At NEB, we have observed EDTA has a strong influence on the 230 nm absorbance and a minute concentration reduction of free EDTA may lead to a higher than usual A260/A230 ratio. In some cases, this ratio exceeds a value of 3.0 and is consistent with highly pure samples. In these cases, the elevated value does not have any negative effect on downstream applications
ELUTED DNA DIFFICULT TO DISSOLVE
  Too much input material used
  • If the suggested input amounts are exceeded, the optimal relationship between bead surface area and efficient wrapping of the DNA around the beads is not maintained. This can produce excessive compacting and tangling of the HMW DNA and inefficient resuspension of the DNA during the elution step. Reduce the input amount per the recommendations.
  Binding of DNA to the beads was carried out for too long           
  • Do not exceed the recommended binding time as this will produce excessive compacting and tangling of the HMW DNA and lead to inefficient resuspension of the DNA during elution.
LOW SEQUENCING READ LENGTHS IN LIGATION-BASED NANOPORE SEQUENCING
  DNA is degraded;
  • See “DNA Degradation” section above
  DNA fragment Length is too large
  • For optimal results in ligation-based nanopore sequencing, the DNA fragments should be ~50–250 kb. If pestle homogenization was used, fragment lengths are often up to 500 kb. Needle shear 10–20X with a 26G blunt end needle as recommended in “Considerations and Performance Data for Nanopore Sequencing”)

 

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