Homogenization of High Molecular Weight DNA (HMW DNA) Samples After Elution

After elution, HMW DNA requires further manipulation before use and measurement. Eluted DNA will not be uniformly dispersed and often clumps in certain areas of the tube; the DNA requires time and effort to disperse and return to its natural conformation in solution. Spectrophotometric measurements that are carried out immediately following elution will, therefore, not give an accurate indication of the amount of DNA present. Also, if the DNA is not completely dissolved, it may be detected in the OD measurement as turbidity, which will result in reduced A260/A230 values that may be falsely interpreted as impurity. 

General Approaches

Pipetting with Wide Bore Tips 

When the DNA is eluted from the beads, the bulk phase retains the wrapped conformation it had when attached to the bead. Pipetting up and down with a 200 µl wide bore pipet tip breaks this conformation and facilitates dissolving and dispersion.  

Heat 

Heat will reduce the viscosity of the solution and significantly increase the speed of homogenization of HMW DNA solutions. Below are some additional guidelines when using heat: 

  • Temperatures > 60°C are generally not recommended as they will lead to DNA denaturation and degradation.  
  • Incubation at 56°C, the temperature used for elution, is appropriate only for short incubation times. Do not exceed 30 minutes.  
  • Samples can be incubated at 37°C for several hours safely; DNA integrity will not be affected. 
  • Incubation at room temperature overnight facilitates even homogenization of the DNA. 
  • Incubation at 4°C for days or weeks also facilitates homogenization and relaxation of HMW DNA. 


If samples are incubated at 37°C for homogenization, mixing the samples at low agitation speed (300 rpm) will increase homogenization efficiency.   

Time 

HMW gDNA needs time to relax and homogenize. It is generally not recommended to work with freshly eluted DNA unless significant effort is made to ensure even DNA resuspension. Allowing the sample to relax overnight, or for several days, facilitates homogenization. If possible, it is recommended that XL DNA is extracted several days or a week prior to being needed for downstream applications.

Dilution 

HMW gDNA should ideally be kept at a concentration of 100–200 ng/µl for easy handling and reliable analysis. As such, using 200 µl elution buffer is recommended for samples obtained from > 2 x 106 cells, > 500 µl mammalian blood or > 5 µl nucleated blood. The viscosity of the DNA solutions decreases significantly with dilution. After dilution, samples will require additional homogenization by the procedures described below.   

For Samples Agitated at Maximum Speed

When agitation is carried out at 2000 rpm during lysis, purified DNA solutions should be only moderately viscous. In order to homogenize these samples prior to downstream analysis or use, the following steps are recommended: 

  1. After elution, pipette up and down 5–10 times using a 200 µl wide bore pipette; ensure any clumps of DNA are dispersed.  

  2. Incubate DNA samples for 30–60 minutes at 37°C. 

  3. Pipette up and down 5–10 times again using a wide bore pipette tip. 

  4. (Optional) Briefly vortex; short vortexing will not affect overall DNA size.  

DNA is now ready for use in downstream applications and can be handled with standard pipette tips. If samples are not immediately required for downstream use, incubate the sample overnight at room temperature or at 4°C for further homogenization. 

For Samples Agitated at Low Speeds (XL DNA) 

Samples isolated following agitation with low speeds are extremely viscous and require additional effort to relax and homogenize before analysis and use. When homogenized completely, samples will appear consistent throughout the tube and OD measurements will become more consistent. The following steps are recommended: 

  1. After elution, pipette up and down 5–10 times using a 200 µl wide bore pipette; ensure any clumps of DNA are dispersed.  

  2. Incubate DNA samples for 30–60 minutes at 37°C. 

  3. Pipette up and down 5–10 times again using the same wide bore pipette tip. 

  4. Repeat 1–2 times each day for at least 2 days. 
If quantitation of XL DNA remains challenging following the above steps, needle shearing is recommended prior to spectrophotometric measurement. Assuming enough sample is available, transfer 30–50 µl to a new tube using a wide bore pipette tip. Needle-shear this aliquot with a 26-gauge (26G) blunt end needle connected to a 1 ml syringe until the viscosity is clearly reduced; up to 20 times may be necessary. Avoid pulling up any air into the needle. If the sample is too viscous to enter a 26G needle, use a larger gauge blunt end needle (e.g., 20G).  

Needle Shearing of HMW DNA Extracted from Tissue optimizes DNA fragment size range and improves N50 values in Oxford Nanopore Sequencing 



300 ng of HMW genomic DNA was separated on a 0.75% gel using a Pippin pulse gel system (Sage Science) at the 5-430 kb program. 200 ul of a pooled DNA sample from 3 x 20 mg mouse brain preps was needle sheared up to 30 times as indicated in the figure. M = Lambda PFG Ladder (NEB #N0341). 

 

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