Introduction

DNA sequencing service is  available to all research groups within the Center for Mammaliam Genetics (CMG). The sequencing facilities  comprises of  ABI Prism 377XL and 3130XL Automated DNA Sequencers.

Services

CMG DNA sequencing facilities provide the following services

• The sale of ABI Prism BigDye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase, FS. Kits are for sale as a 10, 25, 50 or 100 reaction and one reaction cost $6.75. Please contact Akilah McDaniel or call 352-392-3054 for purchansing information.
• Loading of DNA sequencing reaction on ABI DNA sequencer, generation of DNA sequencing data file, and troubleshot of DNA sequencing. Average lenght of DNA sequencing is 700 nucleotides. Average turnaround time is 3 working days. The cost for running DNA sequencing reaction is $2.50(For CMG members) or $3.00(For Non-CMG member).
• Analysis of DNA sequencing files using Factura Feature Identification Software. Factura identifies specified vector and ambiguity ranges, rectriction sites, and a specified confidence range. It also identifies multiples base positions with codes described by the International Union of Biochemists (IUB codes) based on a user-defined threshold. No charge for this service.
• GeneScan run. GeneScan allow to size and quantify DNA fragments. This service cost $100 per gel and up to 34 samples or $3.00 for per sample.(can be run in one gel. You can label different DNA fragments with up to three different color flourescent dyes, you can use a fourth dye color to label an internal DNA standard. Please inquire about this service.
• Plese summit your samples using our sample form.
• Bring samples to sixth floor of the Dental Tower Building to room number D6-19(Address: CMG DNA Sequencing Core Facility, 1600 SW Archer Rd, PO Box 100275).
• Data files can be download using CMG FTP server   from our serve (using WS_FTP, your usename and password)  ftp://dnasequencecore.cmg.health.ufl.edu or bring your floppy or Zip disk.

Protocols

Use the following protocols for DNA sequencing:

• You need Adode Acrobat Reader to view and print protocols.
• Automated DNA sequencing chemistry guide.
• ABI PRISM BigDye Terminator cycle sequencing ready reaction.
• ABI PRIMS Dye Terminator cycle sequencing ready reaction.

DNA Sequencers

 The ABI PRISM 377XL and 3130XLautomated DNA sequencer will automatically analyse DNA molecules labelled with any of four different fluorescent dyes. After samples are loaded onto the system's vertical gel system, they undergo electrophoresis, laser detection, and computer analysis. Electrophoresis separation can be viewed on-screen in real-time, and final data can be printed in a variety of formats. This equipment provides a rapid and inexpensive method for deriving DNA sequence from both cloned DNA inserts and PCR fragments.

377XL sequencing chemistries:

• Four types of sequencing chemistry are now available for the ABI 377XL equipment. Taq polymerase Dye Primer chemistry, Dye Terminator chemistry, dRhodamine Terminator chemistry and the latest Big Dye Terminator chemistry. Selection of the correct chemistry for a given sequencing strategy can greatly enhance your results.

3130XL sequencing chemistries:

• Two types of sequencing chemistry are now available for the ABI 3130XL equipment. dRhodamine Terminator

Program to view and edit ABI DNA sequencing files

• Applications for viewing ABI files created by 377 or 3130. Versions for both Mac and PC systems are available.
  • EditView 1.0.1 for Macintosh systems
  • Chromas141-32.exe for PC systems
  • Chromas14.exe. For non 32 bit PC systems

For further information contact the DNA Sequencing Assistant Scientist at 352-392-7835, Jing-Da Shi, M.D.,Ph.D.