Home › Genetics / Genomics › Extraction: High Molecular Weight Genomic DNA From Soils & Sediments l Protocol Preview
Steps
  1. 1 Prepare denaturing buffer with beta mercaptoethanol 00:56
  2. 2 Assemble and chill mortar and pestle 01:20
  3. 3 Add soil sample and denaturing buffer to mortar 01:30
  4. 4 Cover sample with liquid nitrogen 01:43
  5. 5 Grind sample until powdery and homogeneous 01:48
  6. 6 Repeat grinding with additional liquid nitrogen 01:56
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Extraction: High Molecular Weight Genomic DNA From Soils & Sediments l Protocol Preview

Protocol
Difficulty
intermediate

Steps

1
Prepare denaturing buffer with beta mercaptoethanol

Complete the denaturing buffer by adding beta mercaptoethanol to a concentration of 0.5 percent. Prepare the solution fresh or use buffer less than one week old kept at 4 degrees Celsius.

▶ 00:56
2
Assemble and chill mortar and pestle

Obtain a dewar of liquid nitrogen and prepare an autoclaved, pre-chilled mortar, pestle, and spatula for use in grinding the soil sample.

▶ 01:20
3
Add soil sample and denaturing buffer to mortar

Place a two-gram frozen soil sample in the pre-chilled mortar and add one milliliter of the denaturing solution.

▶ 01:30
4
Cover sample with liquid nitrogen

Add liquid nitrogen to the mortar to fully cover the soil sample.

▶ 01:43
5
Grind sample until powdery and homogeneous

Use the pre-chilled pestle to grind the sample until the soil particles appear powdery and homogeneous and the sample begins to melt.

▶ 01:48
6
Repeat grinding with additional liquid nitrogen

Add more liquid nitrogen to the mortar and repeat the grinding step to ensure complete homogenization.

▶ 01:56

🚨 Failure Case Library (6) + Submit your own case

critical
DNA Fragmentation and Size Reduction
Extracted DNA shows reduced fragment size on gel electrophoresis or pulse-field gel analysis. UHMW DNA appears degraded with smaller molecular weight distribution than expected.
💡 6 · ✓ 6
severe
Low DNA Yield from HMW Extraction
Recovered DNA concentration is significantly lower than expected based on input amount. Spectrophotometric measurements may show unexpectedly low concentrations for UHMW DNA samples.
💡 6 · ✓ 6
severe
Protein Contamination in Purified DNA
Purified DNA shows poor A260/A230 ratio or visible turbidity. Solution may appear cloudy due to protein contamination, affecting downstream applications.
💡 6 · ✓ 6
severe
Low Yield from Tissue Samples
DNA recovery from tissue significantly lower than expected, particularly from muscle, brain, or metabolically active organs. Upper phase transfer yields less DNA than anticipated.
💡 5 · ✓ 5
moderate
Eluted DNA Difficult to Dissolve or Resuspend
DNA pellet or eluate shows poor solubility with visible aggregates or turbidity. DNA remains undissolved after standard elution protocol, requiring extended incubation.
💡 5 · ✓ 6
moderate
Inaccurate Cell Count Leading to Protocol Mismatch
Unexpectedly high or low DNA yield that does not correlate with estimated cell count. Certain cultured cells appear clumped making accurate counting difficult.
💡 4 · ✓ 5
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