DNA Sequencing Facility


DNA Sequencing Facility, Department of Biochemistry
February, 2006
Director:  Charles L. Woodley, Associate Professor of Biochemistry

 

 The DNA sequencing facility uses a LiCor 4000L automated DNA sequencer.  This sequencer is optimal for small sequencing projects in which a few DNA must be sequenced over a length of 1,000 nucleotides.  This is achieved by the use of proprietary primers (currently $114 each) and a traditional slab gel electrophoresis format. This system has the ability to obtain long reads (25-30% longer than capillary gel electrophoresis units). However, projects that require sequencing of a large (>50) number of samples, or SNP (single nucleotide polymorphism) analyses, are best performed on a high through-put capillary electrophoresis system where standard, inexpensive sequencing primers can be utilized.  Scientists at UMMC who need these capabilities now send their DNA samples off Campus for analysis.

      Anyone at UMMC, or those with a history of collaborating with UMMC faculty may make use of the DNA Sequencing Facility. Those scientists interested in the process are welcome to participate in any aspect of the sequencing of their DNA samples. With the current LiCor instrumentation, the facility is capable of processing ~1,250 samples per year.  The facility is currently running at 75% of this capacity. 

General services offered:

  • Sequencing of DNA from either plasmid preparations or PCR products.
  • High resolution polyacrylamide gel electrophoresis with subsequent staining with silver, ethidium bromide or SYBR Gold® (for DNA) and SYBR Orange® (for protein).
  • Quantitation of DNA using a fluorescent dye-binding assay.
  • Culture of bacterial cells with the subsequent preparation of either genomic or plasmid DNA.
  • Primer design for PCR or DNA sequencing reactions.
  • Cloning of PCR products for use in sequencing reactions.
  • Provide, on a limited basis, software for DNA analysis and assembly of DNA fragments into contigs.  This software includes Chromas®, Chromas Pro®, and GeneTool®.  Software analysis may be performed either by the DNA Sequencing Facility or by the end user.  Depending upon the software package used, the analysis may be performed on a computer within the DNA Sequencing Facility or over a network connection on the client’s laboratory computer.  Due to limitations of the UMMC firewall and network, this software is not available off campus.
  • The DNA Sequencing Facility maintains a FTP site from which clients may retrieve, at any time, their current or past sequencing data.  The data is available in several formats, including text based files, a TIF file containing the actual fluorescent profile of the DNA bands eluting from the gel, and as SCF files containing pictorial representations of the sequence profile for each base (A,T,G,C).  This FTP site also contains information related to our protocols and pricing, as well as DNA sequences of cloning vectors and available proprietary sequencing primers. To insure that historical sequencing data will always be available to the client, all data is backed up daily to three computers with a total of five hard drives. The FTP site is available only to computers on campus.  Individual client’s sequences are password protected.  For those who are off campus, sequencing results are provided via e-mail.
  • The DNA Sequencing facility maintains four PCR units available without charge to anyone at UMMC.  These units accommodate either 0.2 or 0.5 mL PCR tubes.  One of these units allows the use of a standard 48-well PCR plate.
  • All procedures are performed on a cost of materials basis.  There are no fees for use of instruments or labor charges.

Currently, numerous laboratories on campus must send their DNA sequencing off-campus since the DNA Sequencing Facility is limited by the available technology. An upgraded system consisting of a capillary gel electrophoresis system, coupled with a minimum of automation, would allow for the rapid completion of large sequencing projects. Specifically, the following items would form a nucleus for an improved, updated system: 1) a high throughput capillary DNA sequencer; 2) a 96-well PCR instrument; 3) additional computing capacity as well as DNA analysis software and, 4) a secure FTP or WEB site where clients could obtain their sequencing results.

 

Pricing Schedule for UMC DNA Sequencing Facility
February 7, 2006

General Policies:

  • The cost of oligonucleotides and other consumables purchased for specific projects are billed at invoice cost.  The current price of labeled primers from LiCor is $114.
  • The costs of other supplies are a part of the billed price of the procedure in which the consumables are utilized.
  • Failed reactions or procedures will not be billed unless it is the result of materials supplied by the laboratory contracting the work.
  • Laboratories supplying materials or labor will receive a reduction in billed cost commensurate the materials/labor supplied.

Procedure

Price

Sequencing gel, prorated, $1.00/set

$10.00

Agarose gel /w photograph

$10.00

DNA Concentration by fluorimetry, per sample

$0.20

Primer Design

$5.00

PCR Analysis, up to 10 reactions, /w gel & photograph

$10.00

Plasmid DNA mini-preparation, each 5 mL sample

$4.00

Plasmid DNA midi-preparation, each 20 mL sample

$8.00

DNA purification/cleanup

$1.00

Sequencing Rx., four base set

$12.00

Clone PCR product or restriction fragment

$35.00

DNA sequence data analysis, per set

$5.00