Sanger DNA Sequencing

The CHGI uses standard automated sequencing methods on our 3730xl Applied Biosystems Genetic Analyzer which generates DNA sequencing read lengths up to 700 bases.

As of October 3, 2022, results will be returned every Monday, Tuesday, and Wednesday.

Results are posted on our secure web server. DNA sequencing files are available in both .txt and .ab1 formats.

If you have trouble, please don't hesitate to contact us. We offer a variety of sequencing troubleshooting protocols, and we are here to help!

DNA

The quality of DNA in a reaction can affect the performance of the DNA Analyzer. When preparing DNA templates, it is critical to submit them in nuclease-free water only, and avoid the following:

• EDTA
• residual salts
• proteins
• residual detergents
• residual RNA

The presence of contaminants can interfere with the sequencing reaction, capillary electrophoresis, and electrokinetic injection. Your current template purification methods may have to be modified to remove these contaminants.

Primers

For best success, primers must:

• be gel purified
• have a Tm between 52°C and 74°C
• not form dimers
• not contain hairpins

Economy Service (DNA and primer premixed)

For each economy reaction prepare the following in one 200 μL tube:

• DNA template
  • 100 ng per 1000 bp for small DNA templates (plasmids, amplicons)
  • 1 μg for large DNA templates (BACs, PACs, YACs, cosmids, and fosmids)
  • 2-3 μg for genomic DNA
• primer
  • 1 μL of 5 μM for plasmid and amplicon sequencing reactions
  • 2 μL of 5 μM stock for large, genomic, or difficult sequencing reactions
• nuclease-free water to a final volume of 12 μL

Full Service (DNA and primer submitted separately)

For full service reactions, please submit separate tubes for each of the following:

• DNA template
  • PCR products: 10 μL at 50 ng/μL
  • plasmids: 10 μL at 200 ng/μL
  • large DNA (BACs, genomic): 10 μL at 500 ng/μL
• primer
  • 5 μL of primer at a concentration of 5 μM