Introduction

Georgetown University has a shared, fee-for-use DNA sequencing and PCR fragment sizing "core" laboratory. This service utilizes an ABI 3100 Genetic Analyzer (a fluorescence based DNA analysis system utilizing capillary electrophoresis). This service is available to all research laboratories on both the Medical Center and Main Campuses.

The service provides high quality results, fast turn-around times and no minimum number of reactions. In addition, the instrument managers can provide detailed and personalized technical support tailored to specific projects.

Prices and Services

DNA sequencing

Sequencing reaction carried out by sequencing core personnel (template and primer provided by investigator) and instrument run (includes big dye reagent)

$7.50 per reaction

Investigator-prepared DNA sequencing reaction instrument run only (does not include big dye reagent)

$4.25 per reaction

Full plate (48 to 96 samples run at one time) of investigator-prepared DNA sequencing reactions instrument run only (does not include big dye reagent)

$200 per plate

DNA Fragment Analysis

Investigator-prepared fragment analysis runs (up to 5 dye colors depending on primer label chemistry)

$4.25 per reaction plus cost of GeneScan size standard (about $0.25 per reaction)

Full plate (48 to 96 samples run at one time) of investigator-prepared fragment analysis runs instrument run only

$200 per plate plus cost of GeneScan size standard (about $0.25 per reaction)

THESE PRICES ARE SUBJECT TO CHANGE AT ANY TIME. Contact the instrument managers to verify current prices.

Questions? Want to use the service? Need technical advice? Contact the instrument managers

Caroline Sheridan

Department of Biology
Reiss 703
(202) 687-9854

or

Md Naimul Islam

Lombardi Comprehensive Cancer Center

Macromolecular Analysis Shared Resource
E401 New Research Building

Phone: (202) 687-7776

http://lombardi.georgetown.edu/research/resources/macromolecular.htm

Purchasing Big Dye v3.x for Sequencing

We purchase Big Dye version 3 in large quantities and sell 100 reaction aliquots to users. Big Dye is substantially less expensive when purchased in 5000 or 1000 reaction quantities, compared to 100 reaction quantities. Buying Big Dye from us will save you money. To purchase Big Dye, provide us with a cost center number and we will JV the cost to your account. Contact the instrument manager for current pricing.

Obtaining Sequencher software

Users of the instrument can utilize a group license for Sequencher, an application to align and edit sequence data. GeneScan is available for fragment size data. You can also download sample files to your computer after runs are complete. Contact the instrument manager to get instructions and all the stuff you'll need.

Acknowledgments

Purchase of the 3100 Genetic Analyzer was supported by an award from NSF Division of Biological Instrumentation to M. Hamilton, H. Elmendorf and J. Neale of the Department of Biology (DBI-0100061). Cost-sharing funds were generously provided by Georgetown University College and Graduate Deans and the Department of Biology.

Publications arising from the use of the instrument should acknowledge these sources of support. We suggest using the following:
"Instrumentation for DNA sequencing was supported by an award from NSF (DBI-0100061) and Georgetown University."
or
"Instrumentation for DNA fragment analysis was supported by an award from NSF (DBI-0100061) and Georgetown University."

Users should notify the instrument manager of published papers that acknowledge use of the instrument.

Protocols

Download Sequencing_protocol.pdf for those labs carrying out DNA sequencing reactions. This version was posted February, 2008.

Links & Useful Information

Applied Biosystems Publications
Go to the ABI Literature and Application Notes site. Then using the search function, type in the document title below, click the "All" radio button, then click Search. (Usually using just the first few words of the document title is sufficient.)

- Sequencing Chemistry Guide
- Big Dye Terminator v3.1 Cycle Sequencing Kit Protocol
- Sequencing Difficult DNA Templates
- Automated DNA Sequencing Chemistry Guide
- Precipitation Method to Remove Unincorporated Dye Terminators

Other Publications

The QIAGEN Guide to Template Purification and DNA Sequencing. Go to the Qiagen website literature section and type in the title.

Web sites

Roswell Park Cancer Institute
An incredibly good web site with information on sequencing basics, including details on preparing plasmid prep and PCR product templates, interpreting chromatograms (with examples), and troubleshooting data.

University of Leicester
Troubleshooting guide: examples of how raw and analyzed data should look (based on ABI 377).

University of Michigan
For useful info on this page, look under the heading "How to Prepare Samples for Sequencing." They have quite detailed information about recommended amounts of DNA. Also look under the heading "About Your Results" and "In case of Problems" for interpreting chromatograms and troubleshooting.

Wake Forest University
Sample preparation and detailed table of recommended quantity of PCR Product

Robarts Research Institute
Sample preparation, amount of contaminants tolerated, data interpretation.

University of Chicago
A guide to preparing plasmid preps and PCR product templates for sequencing, and interpreting chromatograms (with examples).

Duke DNA Analysis Facility
Sample preparation, data interpretation and troubleshooting.

University of Iowa
Detailed protocols for preparing plasmid prep and PCR product templates for sequencing.