Sanger Sequencing (Capillary Electrophoresis)

1. Sanger Sequencing Facility Overview

Create a GCC Biotech sequencing account

The GCC’s Sanger Sequencing Facility provides high-quality capillary-based fluorescent sequencing on ABI 3500 Genetic Analyzer. For large scale products, a second ABI 3730XL can be used. This instrument is usually held in reserve for SNP and microsatellite genotyping.

The facility routinely sequences:

  • Plasmids
  • PCR products
  • Genomic clone ends (cosmids, BACs, PACs, phage)
  • Primer Walking

Sanger CE read lengths are typically 700 bases, with a usual turnaround of three days. The facility currently averages about 3,500 sequence reads per week.

2. Sanger Sequencing Pricing

The facility charges $5.30 per successful read. BAC, LAMBDA, Cosmid and Fosmid reactions will remain at $12.00 each. Failed reads will be charged provided the control from the same sequencing run passes our QC metrics. We accept Visa/MasterCard/AMEX or institutional purchase orders for payment. Credit Card Payment Registration

3. Sanger Sequencing Sample Submission Guidelines

Submit samples using the on-line DNA sequencing Facility GCC Server. Instructions for creating an account, placing orders, etc. can be downloaded. Please note that the individual sequencing data will be unavailable from GCC Server every 15 days from the date of upload.

Samples must be submitted containing 10.0 µl purified template DNA (at the appropriate concentration - see "DNA Template Concentration" instructions below). The entire sample volume will be used for the sequencing reaction. For the best results, please prepare your DNA in water, not TE or buffer. EDTA and salts can interfere with the sequencing enzyme.

If you are using a custom primer, samples must be premixed with template and primer in one tube in order to be processed. The primer must be added at a concentration of ~50ng (~5 pmols) in 0.7-1µl volume, for a total sample volume submitted to the sequencing facility of 7.7 µl. Be sure to only add ONE primer to each tube.

If you are using a universal primer (see GCC universal primer list), the facility will add the primer to your samples. In this case, the volume submitted to the sequencing facility should be 7.0 µl.

DNA Template Concentration

The quantity of DNA template depends on the size of the template you are sequencing.

  • Plasmids - 200-300ng in 7µl
  • PCR product (>4kb) - 150-200ng in 7µl
  • PCR products (2-4kb) - 100-150ng in 7µl
  • PCR products (1-2kb) - 50-100ng in 7µl
  • PCR products (<1kb) - 50ng in 7µl
  • PCR products (<500bp) - 20ng in 7µl
  • PCR products (<200bp) - 10ng in 7µl
  • BAC/Cosmid DNA/Phage DNA - 600-800 ng in 12 ul with 2 ul primer for a total volume of 14 ul
  • Genomic DNA - Please do not submit this (treated as project)

The quality of your sequencing reaction is directly related to the quality of your template DNA. Please follow our guidelines carefully to maximize the chance of success of your sequencing reaction.

4. Sequencing Resources

Electropherogram viewers are freely available for Microsoft Windows, MacOS Classic, and MacOSX from these sites. GCC does not endorse or support any of this software.

FINCHTV TRACE VIEWER - Microsoft Windows, MacOSX, Linux, Solaris
CHROMAS LITE CHROMATOGRAM VIEWER - Microsoft Windows
APPLIED BIOSYSTEMS SEQUENCE SCANNER SOFTWARE - Life Technologies