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Sanger Sequencing on the 3730

The ABI PRISM® BigDye® Terminator v3.1 Cycle Sequencing Kit is used in all Sanger sequencing reactions, the ABI Big Dye® XTerminator™ Purification Kit is used to remove excess dye terminators after the reaction, and the reactions are then run on the ABI Prism 3730 DNA Analyzer. This yields 800bp, with 700-730 bases of Q>20 if the template DNA was of good quality. A pGEM standard is run on each plate to validate the reactions.

NGC checks the data from all plates and if any problem appears to be an instrument or chemistry failure, the plate will be rerun at no charge. If the client wishes samples to be rerun, NGC will rerun them. If there is no improvement, then the client will be charged for the run, but if the sample gives a better result – an improvement of more than 100 in the Q>20 value – then NGC will not charge for the rerun. If NGC sees an obvious problem with a sample, eg: poor priming, insufficient template DNA, high salt concentration, G/C rich region, or double priming, then we will inform the researcher and suggest remedies.

Processing of each sample takes ~8 hours. The average turnaround time is 1 to 2 business days after the samples have been received.

Sanger Sequencing Sample Submission Guidelines

Downloading Your Sanger Sequencing Data

Samples are analyzed in 96-well format on an ABI Prism 3730 DNA analyzer and the data is transferred to our dnaTools server for DNA sequence sample management. Data can be accessed and downloaded from the dnaTools server.  The server will calculate the Phred Q>20 values for all sequences and these can be downloaded and viewed under the Job Quality Report link on the dnaTools server.  The sequence information or the chromatograms can be viewed or downloaded from the dnaTools server.  Downloads can be either individually or by order number.  For more information, please view our dnaTools server guide: How to use dnaTools for Sanger Sequencing .  The chromatograms will have the .ab1 file extension on them.  Other options will contain different file extensions depending on the data downloaded.  For example: Text = .seq files and Phred quality scores = .qual files which can be viewed in Microsoft Word or Excel.

To view chromats you must first download the .ab1 files and then open the .ab1 file(s) using software that will allow viewing of .ab1 files. If you have FinchTV, that will allow you to open and view the files; however, Finch TV is no longer supported or available to download. Current freeware options include:

  1. Technelysium’s Chromas is a free, simple, easy-to-use viewer for Sanger sequencing chromatograms. Download your copy of Technelysium viewer.  
  2. SnapGene offers freeware for Windows, macOS, Ubuntu Linux, or Fedora Linux devices to view Sanger sequencing chromatograms. Download your copy of SnapGene freeware. Download:

For assistance in analyzing your sequencing data please see our Sanger Sequencing - Analyzing Your Data guide.

Additional troubleshooting can be found in the following: