DNA extraction is the foundation of any sequencing workflow. Yet, even experienced labs can make mistakes that compromise DNA quality, yield, or integrity. Whether you are running a diagnostic lab, performing research, or conducting high-throughput sequencing, avoiding these common pitfalls can dramatically improve your results. Let’s dive into the most critical mistakes, why they happen at a molecular level, how to troubleshoot them, and how you can steer clear of them.

Mistake 1: Poor Sample Collection 

Using outdated or incorrect anticoagulants (like heparin instead of EDTA), improper storage temperatures, or delayed processing can lead to DNA degradation. EDTA acts as a chelating agent, binding metal ions (like Mg2+), which are necessary cofactors for nucleases. Without EDTA, nucleases can remain active and degrade DNA. Heparin, on the other hand, can directly inhibit polymerase enzymes used in PCR and sequencing. This not only reduces DNA quality but also introduces fragmentation, which can severely impact sequencing data quality.

How to Avoid:

• Always use EDTA tubes for blood collection for DNA extraction. Heparin can inhibit downstream PCR and sequencing reactions.

• Store blood samples at 4°C if processing within 24 hours or freeze at -80°C for longer storage.

• Avoid multiple freeze-thaw cycles, which can cause DNA shearing.

Mistake 2: Inadequate Lysis of Cells

Insufficient lysis due to low detergent concentration, improper lysis buffer composition, or insufficient incubation time can leave cells intact and lead to poor DNA yield.

How to Avoid:

• Use a lysis buffer optimized for blood samples, containing SDS or other effective detergents.( can be chaotropic agents too- so can write containing effective lysing agents like detergents or chaotropic salts

• Ensure proper mixing of the buffer with the sample.

• For high cell counts, increase lysis buffer volume proportionally. Optimize lysis incubation time based on sample type.- optimal tie- based on the sample type and based on the SOP- recommended.

Mistake 3: Suboptimal Protein Precipitation 

Proteins are denatured by organic solvents like phenol-chloroform or by high salt concentrations. Insufficient separation can leave protein contaminants in your DNA, affecting downstream applications like PCR and sequencing.

How to Avoid:

• Optimize protein precipitation using cold isopropanol or ethanol.

• Use proteinase K for enhanced protein digestion.

• Assess protein contamination using A260/A280 ratios.

Mistake 4: Incomplete DNA Purification

Traces of salts, phenol, ethanol, or other contaminants can interfere with polymerase activity and can inhibit downstream enzymatic reactions, leading to poor sequencing quality.

How to Avoid:

• Perform an additional wash step with 70% ethanol during DNA purification

• Test DNA purity using A260/A230 ratios for salt contamination.

Mistake 5: Using Suboptimal Elution Conditions

Elution in low-pH buffers ( lower than pH 6)  can promote acid hydrolysis of DNA, causing degradation.

How to Avoid:

• Use a buffer with a neutral pH (7-8) for elution.

• If using TE buffer, ensure the EDTA concentration is appropriate for your downstream applications.

Best Practices checklist for Blood DNA Extraction for Sequencing workflows:

1. Use Appropriate Blood Collection Tubes
   Collect blood in tubes with suitable anticoagulants like K2 EDTA to preserve DNA integrity and prevent clotting.

2. Process Samples Promptly or Store Properly
   Process blood samples within 1–2 days at 4°C or freeze at -70°C to -80°C for longer storage. Avoid repeated freeze-thaw cycles by aliquoting samples.

3. Optimize Sample Input Volume
   Use the recommended blood volume (e.g., ~200 µL for whole blood) to avoid overwhelming extraction chemistry and ensure optimal yield and purity.

4. Efficient Cell Lysis with Proper Mixing
   Use appropriate lysis buffers and Proteinase K digestion. Vortex gently during lysis to improve cell disruption and DNA release.

5. Remove Red Blood Cells (RBCs) Effectively
   Apply RBC lysis buffers or density gradient centrifugation to eliminate non-nucleated RBCs that interfere with DNA purity.

6. Thorough Washing Steps to Remove Contaminants
   Perform multiple wash steps to eliminate proteins, hemoglobin, salts, and other inhibitors that can affect downstream enzymatic reactions and sequencing quality.

7. Avoid Overdrying DNA Pellets
   Dry DNA pellets just enough after alcohol precipitation; over-drying makes resuspension difficult. If needed, gently warm pellets at 50–55°C to aid dissolution.

8. Use Fresh, Properly Stored Enzymes and Reagents
   Aliquot enzymes like Proteinase K to prevent activity loss due to repeated freeze-thaw cycles. Prepare reagents fresh or within their validated usage timeframes.

9. Maintain a Clean, DNase-Free Work Environment
   Use DNase-free consumables and separate DNA extraction areas from PCR or library prep to prevent contamination and degradation.

10. Assess DNA Quality and Quantity Before Sequencing
    Measure DNA concentration and purity (A260/280 and A260/230 ratios) and check integrity via gel or capillary electrophoresis to ensure suitability for sequencing.

Following these best practices helps ensure high-quality, high-purity DNA extraction from blood, which is critical for reliable and efficient sequencing workflows such as whole-exome or whole-genome sequencing.

At Cambrian, we’ve fine-tuned our blood DNA extraction kits to deliver consistently high-quality DNA for your sequencing workflows. Learn more.