Sanger Sequencing Service

DNA sequencing is performed on an Applied Biosystems model 3730xl DNA Analyzer or Applied Biosystems model 3130xl Genetic Analyzer. Sequencing is available on PCR products, plasmids and cosmids.

Submission Instructions

In order for automated DNA sequencing to be successful, two factors are crucial:

1. The template DNA must be of high purity
2. The template DNA must be accurately quantified

Failure to meet either criterion may result in poor or no useful sequence data.

Each 0.2 ml 8-strip PCR tube should contain the template DNA and primer combined, in a final volume of 21 µl. 96-well plates are acceptable for orders between 48-94 samples.

Follow the instructions below for required DNA amounts per reaction based on the template type submitted.

Required Consumables

• 0.2 ml PCR strip tubes (Axygen; Cat# PCR-0208C) Krackeler #383-PCR-0208C-PK (for 125 strips of 8 tubes)
• 0.2 ml PCR strip caps (Axygen; Cat# PCR-02CPC) Krackeler #383-PCR-02CPC-PK (for 125 strips of 8 caps)
• 96-well PCR plates (Axygen Cat# PCR-96M2-HS-C or equivalent) The strip caps listed above are compatible with these plates.

PCR Products

1. PCR products must be supplied in an amount determined by the fragment length. This is equal to the fragment length multiplied by 0.1. For example, a 500 bp PCR product sample should contain (0.1)(500) = 50 ng total DNA.
2. 10 pmol of primer, whether universal or unique.
3. Additional ddH₂O, up to a final volume of 21 µl

Plasmids

1. 1 ug of template.
2. 10 pmol of primer, whether universal or unique.
3. Additional ddH₂O, to a final volume of 21 µl

Cosmids

1. 2 ug of template.
2. 20 pmol of primer, whether universal or unique.
3. Additional ddH₂O, to a final volume of 21 µl

Service Requests or Submission Instructions

To request Sanger sequencing services from the AGT Core, please email_[1] or call 518-486-4099.