Bi-directional Sequencing
Your Industry, Our Focus
Reliable Sanger Sequencing for DNA and Plasmid Isolation
Eurofins Genomics offers bi-directional sequencing up to 1500 bases, including DNA/plasmid isolation. You can provide tissue samples, genomic DNA, purified plasmid, or PCR products for sequencing.
Compliant with ISO 9001-2015, Eurofins Genomics is GLP and GMP accredited. For more information or assistance with your sequencing needs, please contact us.
Sample Preparation and Shipping Guidelines
Template - DNA Information
- Plasmid DNA: Appropriate DNA concentration is needed to perform the QC as well as the sequencing run (Please check sample preparation guidelines).
- PCR Product: Indicate on the order form. Sample concentration adjustments are based on product size.
- DNA Concentration: Provide accurate concentration. Low concentrations may lead to QC failures.
- Special Conditions: Note if your DNA sequences contain high GC content, repeats, or poly A regions for optimized sequencing conditions.
Sample Name Clarification
- Consistency: Ensure sample names on tubes and order forms are identical.
- Uniqueness: Use different template names for each order to avoid confusion.
- Length: Keep sample names concise. Combined template and primer names over 28 characters will be truncated.
- Labelling: If using screw-on caps, label both the tube and the cap.
Shipping
- Protection: Use proper precautions to prevent mechanical damage to tubes containing DNA or PCR products.
Sample Submission
For our Value Read service, submit samples correctly to ensure accurate results.
- Containers: Use 1.5 ml safe-lock tubes for templates and primers or 0.2mL PCR products. Place 0.2mL PCR tubes in 1.5ml Eppendorf tubes and wrap them with Parafilm.
- Labelling: Use a water-resistant marker for additional labelling.
Sample Preparation Guidelines
Use the following concentrations and volumes for your samples:
|
Sample Type |
Product Length |
Sample Conc. |
Sample Vol. |
|
Plasmid DNA |
--- |
50-100 ng/µl |
20 µl |
|
Purified PCR Products |
150-300 bp |
50 ng/µl |
20 µl |
|
Purified PCR Products |
300-1000 bp |
50 ng/µl |
20 µl |
|
Purified PCR Products |
1000-3000 bp |
50 ng/µl |
20 µl |
|
Unpurified PCR Products |
150-300 bp |
50-70 ng/µl |
20 µl |
|
Unpurified PCR Products |
300-1000 bp |
50-70 ng/µl |
20 µl |
|
Unpurified PCR Products |
1000-3000 bp |
50-70 ng/µl |
20 µl |
Ensure accurate template concentration via agarose gel or photometer.
Premixed Samples (Template and Primer Mixture)
- Template: 15 µl purified DNA at the specified concentration.
- Primer: 2 µl of primer at 10 pmol/µl (10 µM).
- Total Volume: Ensure the premixed sample totals 17 µl.
Sequencing Primers
Optimum Primer Conditions
- Composition: Primers should not contain phosphorylation or fluorescent dyes.
- Length: 16-25 bases.
- Melting Temperature (Tm): 50 - 62°C.
- GC Content: 35-60%, ideally with one G or C at the 3' end and no more than two 3' Gs or Cs.
- Sequence: Avoid more than three identical bases in a row.
Primer Concentration and Volume
- Concentration: 10 pmol/µl per sequencing reaction.
- Volume: Each primer should have a total volume of 15 µl. An additional 5 µl is required for each extra sequencing reaction.
Frequently Asked Questions ( FAQ's )
What is bi-directional Sanger sequencing?
Bi-directional Sanger sequencing involves sequencing DNA fragments in both forward and reverse directions to ensure accuracy and comprehensive coverage of the target sequence.
How does Sanger sequencing work?
Sanger sequencing works by synthesizing DNA strands from a single-stranded template, incorporating fluorescently labelled dideoxy nucleotides that terminate the chain, and then separating the resulting fragments by size to determine the DNA sequence.
What are the applications of Sanger sequencing?
Sanger sequencing is used for various applications including mutation detection, microbial identification, sequence verification, and genetic research.
Why is bi-directional sequencing more accurate?
Bi-directional sequencing provides data from both directions of the DNA strand, which helps to confirm the sequence and identify any errors or ambiguities that might arise from single-direction sequencing.
What is the difference between Sanger sequencing and next-generation sequencing (NGS)? Sanger sequencing is a golden standard method for the short read generation of DNA fragments with high accuracy, while NGS is a high-throughput method that sequences millions of fragments simultaneously, offering greater speed and depth but with a different error profile.
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