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DNA Sequencing



The Biopolymer/Genomics Core Facility offers dye-terminator-based automated DNA sequencing, supported by an Applied Biosystems model 3730XL 96-capillary high-throughput sequencer. The model 3730XL can run 96- or 384-well format plates, and, depending on the desired read-length, can assay at least 12 - 96-well plates per 24 hour period, running unattended. This instrument offers greatly improved efficiencies in labor and consumables, allowing us to decrease user fees for this service and decreasing turn-around time.

Important Considerations

  • Accurate concentrations need to be provided.
  • A gel picture can be substituted for PCR product concentrations.
*PLEASE NOTE AMOUNT LOADED PER LANE*
  • All DNA and Primers must be in Water and clearly labeled.
  • Provide accurate email address to have your data emailed.
  • Indicate whether you are submitting plasmid or PCR product
  • Check the boxes listed under Service Requested
  • When requesting Complete Single or Complete Double Stranded Sequence, make sure to indicate the length of your insert or PCR product
 
Sequencing reactions are performed using a variety of thermal-cyclers, including Applied Biosystems models 9600 and 9700 and an MJ tetrad, with four 96 well blocks. The facility offers flexible alternatives for our users. Investigators provide the facility with DNA, and any custom primers (or sequences of primers to be synthesized), and the facility will perform and run the sequencing reaction, and provide the investigator with edited text files electronically.

Alternatively, investigators can perform their own sequencing reactions and submit them as 'ready to run' at a significantly discounted rate. Those investigators who wish to take advantage of this alternative service can purchase aliquots of the dye-terminator reaction mixture from the facility at our reduced bulkrate.

The facility has extensive experience in sequencing DNA from a variety of sources including plasmid, PCR products, bacteriophage, BACs and even genomic DNA. An added benefit to having provided DNA sequencing services for over 15 years is that the facility now stocks primers that can be used on virtually any commercially available vector, eliminating the need for repeated synthesis of these sequences. Turn-around time is often 24 hours and every attempt will be made to achieve this timeline.

Data from a full plate of 96 samples submitted as 'ready to run' can be generated in as little as 2-3 hours, depending on the backlog and desired read-length. Projects involving large numbers of samples (multiple 96-wellplates) are handled on a case-by-case basis. Methods are developed to optimize large projects so as to minimize the cost of reagents needed to complete the work-scope.

(Genescan) For fragment analysis, samples must to be submitted in a 96 well tray. Water must be added to any empty wells. Failure to submit a full plate may delay you receiving your data.

Primer walking of large DNA fragments is offered in the facility, although some investigators prefer to analyze sequence data and design their own primers to continue sequencing. For those who wish, the software Primer Design (Applied Biosystems) is available in the facility for their use. The facility utilizes Sequencher software to analyze and assemble large constructs. Many investigators who routinely sequence large DNA fragments have purchased their own copies of Sequencher, allowing easy transfer of data from the facility to the investigator.

High-throughput genomic sequencing platforms are available through the Genomics Resource Center (GRC), a research resource of the University Of Maryland School Of Medicine’s Institute for Genome Sciences. More information on the platforms available and services provided can be found at the GRC web site.

IMPORTANT NOTE

We will no longer be providing printed copies of the chromatograms. If you would like to view and print your chromatogram, you can download freeware from Applied Biosystems to view and print chromatograms. (
CHOOSE DOWNLOAD OF SEQUENCE SCANNER SOFTWARE v1.0).

For more information or questions, please contact Kevin Rossomando.

The cost for DNA sequencing is:

Full Service
(Core does the sequencing reaction, clean-up, run the reaction and provide data)

  • $8.00 per reaction for single reactions
  • $600 per 96 well tray
Ready To Run Samples
  • $3.00 per sample (Maximum of charge of $75.00 if all submitted at once)
  • $75 per 96 well tray

Cost for Primer design and synthesis:

  • Current price per base for synthesis at 40nMole without purification (currently $0.40 per base).

Biopolymer Sequencing Policies

  1. The required Biopolymer/Genomics Core facility DNA Sequencing order form must be submitted when samples are dropped off. Failure to fully complete the DNA sequencing form may result in delay of results.
  2. If the sequencing data is of poor quality and the root cause is proven to have been caused by the Biopolymer lab. The root cause will be determined by running 1 or 2 of customer samples under the same conditions. Then the Biopolymer lab will repeat the sequencing run at no charge to the customer.
  3. If a sequencing run fails to produce useable data due to high G-C rich content or secondary structures and that information was NOT noted on the Sequencing order form. Then the customer is responsible for paying for any sample re-runs.
  4. If a sequencing run fails due to lack of customer information such as no concentration value, low yield or insufficient volume, then the customer is responsible for paying for any sample re-runs.
  5. If customer follows all submission policies and sequencing run fails when the laboratory used the customers sample and primer. Then an available stock primer is selected by the customer and Biopolymer will run a single sample to troubleshoot at no cost to the customer. If successful, the customer may instruct and pay Biopolymer to re-run their samples using its stock primer.
  6. If customer follows all submission policies and sequencing run fails when the laboratory used the customers sample and Biopolymer supplied the primers. Then Biopolymer will re- run a single sample to troubleshoot at no cost to the customer.
  7. If a sequencing run fails and the Biopolymer lab has exhausted all trouble shooting and communicated with the customer and the customer insists on re-running using the same exact conditions and/or samples. Then the customer is responsible for payment of all re-runs.

Policy effective Date 9/4/2012