Home › Molecular Biology › Extraction Of High Molecular Weight DNA From Microbial Mats l Protocol Preview
Steps
  1. 1 Homogenize microbial mat sample 01:41
  2. 2 Extract microbial cells from mat 00:12
  3. 3 Lyse cells using freeze-thaw and chemicals 00:21
  4. 4 Remove cell debris and RNA 00:29
  5. 5 Extract and concentrate DNA 00:39
  6. 6 Precipitate and purify DNA 00:48
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Extraction Of High Molecular Weight DNA From Microbial Mats l Protocol Preview

Protocol
Difficulty
intermediate

Steps

1
Homogenize microbial mat sample

Mix approximately 30 grams of microbial mat thoroughly using a sterile grinding pestle without grinding until no lumps remain visible in the mixture.

▶ 01:41
2
Extract microbial cells from mat

Separate microbial cells from the homogenized mat background matrix using the prepared sample.

▶ 00:12
3
Lyse cells using freeze-thaw and chemicals

Apply freeze-thaw cycles combined with chemical lysis methodology to break open the microbial cells and release DNA.

▶ 00:21
4
Remove cell debris and RNA

Perform organic extractions and RNase treatment to eliminate cell debris and RNA from the lysate, leaving DNA in solution.

▶ 00:29
5
Extract and concentrate DNA

Isolate DNA from the treated solution using extraction methodology to separate it from other contaminants.

▶ 00:39
6
Precipitate and purify DNA

Perform DNA precipitation and purification steps to obtain high molecular weight DNA of optimal quality and concentration.

▶ 00:48

🚨 Failure Case Library (5) + 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
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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