Sanger Sequencing Support | Eurofins Genomics US

Overview

Eurofins Genomics US provides a comprehensive range of support and services for DNA sequencing, including Sanger sequencing, Nanopore sequencing, and Next Generation Sequencing (NGS). This guide focuses on troubleshooting common issues encountered in Sanger sequencing, a widely used method for DNA sequencing.

Key Services Offered

• Sanger Sequencing

  • Sanger Sequencing Service
  • High Volume Sequencing in Plates
  • Direct Colony Sequencing
  • SimpleSeq Kits
  • GLP and GMP Sequencing

• Nanopore Sequencing

  • Whole Plasmid Sequencing
  • Linear/Amplicon Sequencing
  • Bacterial Genome Sequencing

• Gene Synthesis & Fragments

  • Gene Synthesis
  • GeneStrand Fragments
  • IVT mRNA

Troubleshooting Common Issues in Sanger Sequencing

1. Failed DNA Sequencing Reaction or Dirty Sequence

• Appearance: Messy chromatogram, many "N's", low signal intensity.
• Possible Causes: Low DNA template concentration, wrong/no primer, low-quality prep.
• Treatment: Check DNA concentration with agarose gel, verify primer sequence, fresh prep and dilution.

2. Double Sequence Data

• Appearance: Overlapping peaks.
• Possible Causes: Clone contamination, heterozygous PCR template, two primers added.
• Treatment: Replate bacteria for clean clones, use new PCR primers, verify primer sequence.

3. Noisy Background

• Appearance: Background noise under main sequence peaks.
• Possible Causes: Clone contamination, heterozygous PCR template, improper PCR purification.
• Treatment: Replate clones, redesign primers, check purification protocol.

4. Stuttering After Mononucleotide Stretches

• Appearance: Poor data quality after stretches of the same base.
• Possible Causes: Polymerase slippage.
• Treatment: Sequence in reverse direction, use poly-mononucleotide primer with wobble.

5. Mid Sequence Stop or Drop-Off

• Appearance: Sequence stops or drops off suddenly.
• Possible Causes: Secondary structures, GC/GT rich regions.
• Treatment: PowerRead technology can help.

6. Top-Heavy Sequences

• Appearance: Signal gradually drops off.
• Possible Causes: Excess DNA template or primer, GC/GT rich template.
• Treatment: Quantify DNA template accurately, sequence in reverse direction.

7. Spikes

• Appearance: Sharp, multicolored peaks.
• Possible Causes: Impurity/micro bubble.
• Treatment: Re-run sample if necessary.

8. Early Peak Deterioration

• Appearance: Broad peaks early in the sequence.
• Possible Causes: Salt or impurities from DNA isolation.
• Treatment: Purify DNA with a desalting kit.

9. CRISPR and TIDE Assays

• Note: Notify Eurofins early for sequencing related to TIDE assays to adjust protocols accordingly.

Additional Resources

• Account management, order submission, and tracking via online tools.
• Various products for DNA/RNA oligos, qPCR probes, and sequencing kits.
• Support available through phone and email during specified business hours.

Contact Information

• Technical Support: Phone 1-800-688-2248 ext 1, Email: [email protected]
• Billing and Accounting: Phone 1-800-688-2248 ext 2, Email: [email protected]

Conclusion

Eurofins Genomics US provides robust support for DNA sequencing needs, with an emphasis on troubleshooting common issues in Sanger sequencing. For optimal results, users are advised to prepare samples following recommended guidelines and contact support for any specific issues.