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Science

Sanger sequencing

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illustration of a method of dna sequencing

The Ramaciotti Centre for Genomics isÌýcommitted to providing a quality SangerÌýDNA sequencing service with a fast turnaround time. Our services are flexible and can be customised to meet your needs.ÌýAll sequencing performed by the Ramaciotti Centre for Genomics is carried out in our ISO/IEC 17025 accredited laboratories.

If the Sanger sequencing serviceÌýyou requireÌýis not listed on the service menu below or you need assistance with project design pleaseÌýcontact us.

If you are a returning customer, pleaseÌýsubmit your order through the ±è´Ç°ù³Ù²¹±ô.Ìý

  • Our Core Prep services are an easy optionÌýfor researchers who would like to fast track their project or for those who areÌýnew to Sanger sequencing.

    Core prep

    You submit a mix of purified PCR product and primer, we perform the sequencing reaction and capillary separation.
    Turnaround time: 3-5 days.*

    Core prep with clean up

    You submit unpurified PCR product and primer, we perform the PCR clean up, sequencing reaction and capillary separation. Clean up options include ExoSAp-IT and magnetic bead clean up (plate submissions only). Ìý
    Turnaround time: 3-5 days.*

      Returning customers

      PleaseÌýlog in to ourÌýÌýportal to submit your samples.

      New customers

      Please download our user guides for detailed instructions on how to use our Sanger sequencing services:

      Data delivery

      Sanger sequencing results will be available in Lablinks, under each Sanger project's Results and Documents tab. The following files are available to download:

      • ABI format chromatogram
      • QUAL file
      • SEQ file

      Quality guarantee

      • We include internal controls (pGem) on all sequencing runs to ensure quality data.
      • All traces are manually inspected before data is released.
      • Free re-run service if results do not meet our quality standards.

      Ìý

      *ÌýÌýWorking days are Monday – Friday (excluding Public Holidays). Samples must be received by 12 noon to be included in theÌýday's sequencing run.

    • Our User Prep services are useful for researchers who are hands-on and have access to the required sequencing reagents.

      User prepÌý

      You submit clean, pelleted sequencing product, we perform the capillary electrophoresis. Turnaround time: 1-3 days.*

      User prep plus

      You submit the product of the sequencing reaction, we perform the clean up and the capillary electrophoresis. This service is only available for plate submissions.Ìý
      Turnaround time: 1-3 days.*

      Returning customers

      PleaseÌýlog in to ourÌýÌýportalÌýto submit your samples.

      New customers

      Please download our user guides for detailed instructions on how to use our Sanger sequencing services:

      Data delivery

      Sanger sequencing results will be available in Lablinks, under each Sanger project's Results and Documents tab. The following files are available to download:

      • ABI format chromatogram
      • QUAL file
      • SEQ file

      Quality guarantee

      • We include internal controls (pGem) on all sequencing runs to ensure quality data.
      • All traces are manually inspected before data is released.
      • Free re-run service if results do not meet our quality standards.

      *ÌýÌýWorking days are Monday – Friday (excluding Public Holidays). Samples must be received by noon to be included in theÌýday's sequencing run.

    • 1. Sanger sequencing

      Successful Sanger sequencing is achieved by good template-primer complementary and template-primer ratio. The table below shows the recommended quantity of template to use in a sequencing reaction base on the size of the targeted template . The AB3730 DNA Analyzer is a highly sensitive instrument, consequently you may not need as much template as indicatedÌýin the table. Input amounts may have to be optimised as over-saturation of signal will cause a reduction inÌýdata quality.

      As a starting point weÌýsuggest that clients read ourÌýSanger Sequencing Guide.Ìý

      TemplateRecommended quantity
      PCR - 100 - 200bp
      200 - 500bp
      500 - 1000bp
      1000 - 2000bp
      >2000bp      		1 - 3 ng
      3 - 10ng
      5 - 20ng
      10 - 40ng
      40 - 100ng
      Single-Stranded DNA50 - 100ng*
      Double-Stranded DNA (Plasmid DNA)200 - 500ng*
      Large DNA (e.g. BACs,PACs,YACs,cosmids and fosmids)0.5 - 1.0ug
      Bacterial genomic DNA2 - 3ug

      Table referenced fromÌýDNA Sequencing by Capillary Electrophoresis by Applied Biosystems.

      2. Sequencing guides

      Below are links to resources for general information on Sanger sequencing and troubleshooting your data:

      Ramaciotti Centre Sanger Sequencing Guide

      DNA Sequencing by Capillary Electrophoresis by Applied Biosystems

      Qiagen Guide to Template Purification and DNA Sequencing

      3. Sequencing clinic

      The Centre runs a sequencing clinic whereby clients can book one on one time with a Sanger sequencing expert. PleaseÌýcontact usÌýfor further information or to make an appointment.

      4. Software for viewing and editing chromatograms

      We advise our customers to check the chromatograms manually to ensure correct base calling and genotype calling. There are a largeÌýnumber of programs available, each having itsÌýown specific advantage.Ìý

      • Applied Biosystems Sequencing Analysis 5.1.1 for Windows XP and SeqScape (Sequencing Analysis and alignment) fromÌý.
      • Ìýsoftware for viewing and editing chromatograms (Macintosh OS X, Windows, Linux). Free access.
      • Ìýis a comprehensive suite of tools for analysis of a range of sequencing data. Trial and student discounts are available.
      • Genemapper 5.0 for Windows 7 is available for Microsatellite, AFLP and RFLP analysis fromÌý.
      • Sequence Scanner (Sequencing Analysis) and Peak Scanner (Fragment Analysis)ÌýÌýfor PC. Free access.
        Ìý

      The Centre does not provideÌýsoftware for analysis of sequencing data but can provide anÌýanalysis service if required (conditions and fees apply).ÌýContact usÌýfor further information.
    • Service typeSubmission formatReaction numberPrice/Reaction (ex GST)
      Core Prep

      Tube

      Plate

      Any

      40 - 96

      $11.50

      $9.50

      Core Prep & ExoSAP-IT

      Tube

      Plate

      Any

      40 - 96

      $13.50

      $11.50

      Core Prep & Bead Clean UpPlate40 - 96$13.50
      User Prep

      Tube

      Plate

      Any

      40 - 96

      $7.00

      $5.00

      User Prep PlusPlate40 - 96$7.20

      Included in ourÌýservices:

      • Routine quality checking of all traces.
      • Easy access to your sequencing data.
      • Technical support.
      • Troubleshooting advice.

    • Software for viewing and editing chromatograms

      There are a number of analysis program options available:

      • Applied Biosystems Sequencing Analysis 5.1.1 for Windows XP and SeqScape (Sequencing Analysis and alignment) fromÌý.
      • Genemapper 3.7 for Windows XP is available for Microsatellite, AFLP and RFLP analysis fromÌý.

      The Centre does not provideÌýsoftware for analysis of sequencing data but can provide anÌýanalysis service if required (conditions and fees apply). PleaseÌýcontact usÌýfor further information.

      Other analysis programs (includingÌýfreeware)

      • Sequence Scanner (Sequencing Analysis) and Peak Scanner (Fragment Analysis)ÌýÌýfor PC.
      • FreeÌýÌýsoftware fromÌýÌýfor viewing and editing chromatograms (Macintosh OS 9.x, 1.5 Mbytes).
      • FreeÌýÌýsoftware for viewing and editing chromatograms (Macintosh OS X, Windows, Linux).
      • FreeÌýÌýsoftware for viewing and editing chromatograms (Macintosh OS X).
      • Ìý– Biological sequence alignment editor for Windows 95/98/NT.
      • Ìý– a multiple sequence alignment editor, analyser, and shading utility for Windows.
      • Ìý– http://www.vgl.ucdavis.edu/STRand (Fragment Analysis) for PC.

      Other helpful links and guides

      • Ìý– sequence submission support and software.
      • Ìý– a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA.
      • Ìý– an integrated search and retrieval system that takes information from all of the databases at NCBI.
      • Genographer – The MSU Sequencer Users’ Group, EPSCoR Plant Biotechnology Group, Barley Genetics, Genographer and Barley Production Homepages.