Sanger Sequencing

Competitive Sanger sequencing at the BRF BRF sanger sequencing ABI3730 BigDye

The BRF was established in 1988 and has a long history of DNA sequencing, with a number of instrument upgrades since that time. In 2003 we installed an Applied Biosystems 48-capillary 3730 DNA Analyzer,  which we subsequenctly upgraded to a 96-capillary (3730xl) in 2010, to meet our increasing demand. At the begining of 2021, we said good-bye to our old 3730xl and replaced it with a refurbished unit with a brand-new laser.  

Our facility operates on a ‘cost recovery’. We aim for a turnaround time of 1-3 days from receipt for User Prep samples and 5 working days for Core Prep samples. Internal controls are included in all sequencing runs and our pGem controls routinely produce over 1,000 base pairs of data with a quality score of >20.

Our commitment to our customers is to provide a excellent service, accurate results, fast turnaround times, at low cost.

Services

Sequencing

  • Customer completed reactions (user prep)
  • Sequencing reactions performed by BRF (core prep)

Genotyping 

  • Fragment analysis

Tip

The quality of the data is determined by the quality and quantity of the DNA template and primer that you use. Sample cleanup is critical as large excess Big Dye blobs will saturate the signal and salt will prevent correct electrokinetic injection.

Price for ANU clients

Price is calculated and quoted when you order via dnaLIMS.

Price for external clients

Service

External organisation (non-profit)

User preps (1-95 samples) $4.10 per sample
User preps (96+ samples) $2.60 per sample
Core preps $7.20 per sample
Fragment analysis Please contact BRF

1. Complete our electronic submission form in dnaLIMS

At the BRF, we use the software dnaLIMS to track and process sanger orders and projects. dnaLIMS is a limited-access online software. ANU customers can use the dedicated sample submission computer located in the Sample Drop Off room (2.043).  Customers from CSIRO and UC can access dnaLIMS on computers located at their institutions.

After placing an order, the system generates an order form. Please save an electronic copy and email it to dnaseq@anu.edu.au.

2. Drop off samples

ANU clients with access to JCSMR can drop off their samples in the freezer located at the Sample Drop Off room (2.043).

Clients without access to JCSMR can drop off their samples the freezer located at JCSMR stores at the back of the building.  This is available between 8:30am to 12:30pm and 1:30pm to 4:00pm.

If you are posting samples, please use Express Post bags and wrap samples in bubble wrap to protect tubes. Please address your package to:

The Biomolecular Resource Facility (BRF)
The John Curtin School of Medical Research
Building 131
Floor Dock off Garran Road
The Australian National University
Canberra ACT 2601

3. Get your results

ANU customers will receive sequencing files (.ab1 and .seq) via email. Please make sure to update your email in dnaLIMS as your result will be sent there. CSIRO and UC customers can download results by login onto their dnaLIMS accounts.

 

General

1. The BRF operates on a cost recovery basis. The percentage of subsidization is determined by The John Curtin School of Medical Research.

2. No samples or orders will be processed without an authorised* sample submission/order form and valid charge code. (*Authorised by the signature of the PI/Lab Head, or by granting of electronic access to BRF ordering systems with the authority of the PI/Lab Head).

3. It is a condition of the contract between the ACRF and JCSMR/ANU that "the Foundation (ACRF) is to be acknowledged in all scientific publications which utilise the Facility (BRF) at the Institute (JCSMR)". All work performed in the BRF, whether full service or not, and the use of any BRF resources should be acknowledged in all publications arising from that work. This should be in the “Material and Methods” section of the paper (see examples below). Any further individual acknowledgements are solely at your discretion.

4. A reference to the publication should be sent to the Manager of the BRF once the paper is published (includes theses).

5. BRF collaborations and co-authorship: although the BRF is primarily a service unit, there are instances where BRF staff make significant contributions to either the technical or intellectual input of the project. This should be discussed with the staff member concerned prior to the commencement of the collaboration.

Consumables and data

  1. The customer agrees to cover the cost of any consumables ordered on their behalf if the customer fails to supply the correct quantity and quality of the starting material required for processing. Details of the quality and quantity required are on the BRF website and sample submission form.
  2. Customers are responsible for archiving data generated by the BRF. Data generated by the BRF are solely for the use of the customer and their collaborators. Data is not to be sold to a third party. The BRF shall not be responsible for data output generated from samples that deviate from recommended protocols as requested by the customer.


  3. Upon receipt of consumables and/or data, the customer accepts responsibility for the correct handling, use, storage and disposal of the consumables/data.
  4. The BRF extends no warranties of any kind in respect to the consumables.

  Any consumables sold may have hazardous properties.  The customer agrees to use appropriate caution and safeguards as not all properties are known.
  5. Consumables sold by the BRF shall not be transferred to another party without the written consent of the BRF.  The BRF is not responsible for any losses arising from the use of consumables. The BRF will not be liable to the customer for any loss, claim or demand made by the customer due to acceptance, handling, use, storage or disposal of consumables and/or data by the customer, except to the extent permitted by law when it is the result of willful misconduct on the part of the BRF or its employees.

Acknowledgement in Publications

Examples

Real-time PCR

“Amplifications were performed in 384-well optical reaction plates (Applied Biosystems) with a 7900HT Fast Real-Time PCR System at the Genome Discovery Unit - ACRF Biomolecular Resource Facility, The John Curtin School of Medical Research, Australian National University using SDS 2.4 software to analyse raw data.”

DNA Sanger Sequencing

Amplified PCR products were purified and sequenced on an AB 3730xl DNA Analyzer (at the Genome Discovery Unit - ACRF Biomolecular Resource Facility, The John Curtin School of Medical Research, Australian National University) following the manufacturer's protocol (Applied Biosystems 2002).”

Peptide synthesis

Peptides were synthesized chemically using the 9-fluorenylmethyloxycarbonyl (Fmoc) method on a CEM Microwave-assisted Peptide Synthesizer and purified by one round of C18 reversed-phase HPLC by the Genome Discovery Unit - ACRF Biomolecular Resource Facility at the John Curtin School of Medical Research, Australian National University. As required, the N- or C-terminus, or both, were protected by acetylation or amidation, respectively.”

Tetramer synthesis

Cells were surface stained with APC-labelled tetramers consisting of murine class I MHC molecule (H-2Db), b2-microglobulin and influenza nucleoprotein peptide NP366–374. Tetramers were synthesised at the Genome Discovery Unit - ACRF Biomolecular Resource Facility at The John Curtin School of Medical Research, Australian National University using BirA enzyme synthesized as described (O’Callaghan et al., 1999). [O’Callaghan, C.A., Byford, M.F., Wyer, J.R., Willcox, B.E., Jakobsen, B.K., McMichael, A.J. and Bell, J.I (1999). BirA Enzyme: Production and Application in the study of membrane receptor-ligand interactions by site-specific biotinylation.Anal. Biochem. 266, 9-15.]”