Molecular Core Facility
Services
The facility offers DNA sequencing services to College of Charleston researchers as well as to researchers at other local institutions. A part-time technician, who will collaborate with investigators to optimize procedures and to discuss results, staffs the facility. DNA samples are sequenced using Beckman Coulter dye terminator cycle sequencing quick start kit. Users may choose to perform the cycle sequencing reaction themselves. After cycle sequencing, the fluorescently labeled DNA samples are separated by capillary electrophoresis on a Beckman Coulter CEQ 8000. If low quality results are obtained users may choose to have the reaction repeated and the DNA samples re-run on the sequencer at a reduced rate. However, if poor sequence results are found to have been due to CEQ 8000 instrument error or are otherwise our fault there will not be a charge for this service. Fragment analysis (e.g., microsatellites, AFLPs) is also available. The lab will supply 400 or 600 base pair size standards. Sequence results are provided as text-only files and chromatogram files that are available by e-mail or on user supplied disk.
The features and the services of the Molecular Core Facility can be viewed as a slide show in Adobe Acrobat. In order to view the PDF file, you need Adobe Reader. Click here to get the Adobe Acrobat Reader.
Sample Preparation for Sequencing
High quality template is critical in obtaining good sequence data. It is very important that the DNA is not contaminated by salts, RNA, sequencing primers, ethanol, etc. Beckman Coulter recommends using column purification (e.g., Qiagen QIAquick) or ExoSap enzyme digestion (available through USB). Reliable data have also been obtained from samples purified on a low melting point agarose gel followed by agarase digestion.
The concentration of template DNA also affects the quality of sequence data and is required to determine the amount of template used in the cycle sequencing reaction. Prior to submitting a work order, all templates should be quantified and the method of quantification should be indicated on the work order form. It is strongly recommended that samples be quantified on an agarose gel using a mass ladder.
The amount of DNA required for the cycle sequencing reaction is based on the size of the DNA fragment, at least 33 ng per kilobase of DNA is required. Please note that if you are submitting samples for cycle sequencing you will also need to provide 2 µL of a 5 to 10 µM solution for each primer.
Sample Submission for Sequencing
To submit a sample please complete a work order form and attach or email a photo of the agarose gel showing template purity or indicate the file name that the image was saved as on the BioRad Imager (save to CEQ folder), located at Grice Marine Lab in room 205. Work order forms are available through this web site and in room 210 at Grice marine Lab. Work order forms can be left on the desk or e-mailed to the manager of the molecular core facility. Results will be transmitted by e-mail unless the user provides a CD-R or Zip disk. The data file for each template will be named as follows unless it is requested otherwise: Template Name - Primer Name - Researchers Initials - Date.
Each sample and primer tube should be labeled with the researcher’s initials, date and sample or primer name. Samples can be dropped off at Grice marine lab at 205 Fort Johnson Road, Room 210, during the facilities normal business hours. In the refrigerator there is a designated shelf for samples that need to be cycle sequenced or for those that are ready to be loaded on the CEQ for electrophoresis. Please note that if you are submitting samples for cycle sequencing you will also need to provide 5 - 10 µM solutions of each primer used for amplification. Samples will be discarded after two months unless other arrangements are made.
Samples must be run on the sequencer in multiples of eight. If eight samples are submitted, the sequence results should be available in 3-4 days. AFLP samples must also be run in multiples of eight and results should be available in 1-2 days. If fewer than eight samples are submitted they will be processed as soon as possible; however, researchers may choose to have these samples processed immediately for an additional fee of $1.75 for each sample less than eight. Please note that all samples will be processed as soon as possible; however, during periods of increased use CofC Biology Department faculty and students will have priority. During these times, intermediate and external samples will be processed at least once per week. After submitting samples you may contact the manager of the molecular core facility for the specific date that your results should be available.
Payment
College of Charleston users will receive an IDT each month that should be completed and returned to the manager of the molecular facility.
External users will receive a bill each month that should be paid within two weeks. Payments should be made by institutional check and returned to the College of Charleston’s Treasurer’s Office. Please make the check payable to the College of Charleston and reference account # 350122. You may also contact the Treasurer’s Office at (843) 953-5572 to arrange payment by credit card. The manager of the molecular facility may be contacted with any questions.
Fees Per Sample for Sequencing
- Internal: CofC Researchers (Biology Department)
- *Reaction and Electrophoresis $8
- Electrophoresis only $2.50
- **Re-run failed sequence $6
- AFLP sequencing $3.50
- Intermediate: Non-Biology CofC faculty and Fort Johnson scientists
- *Reaction and Electrophoresis $9
- Electrophoresis only $4
- **Re-run failed sequence $6
- AFLP sequencing $6
- External Researchers
- *Reaction and Electrophoresis $11
- Electrophoresis only $7
- **Re-run failed sequence $6
- AFLP sequencing $8
- *These prices reflect Beckman Coulter’s recommended reaction volumes for cycle sequencing. Smaller reaction volumes may be used at investigator’s request. Contact the laboratory manager for pricing details.
- **Please note that there will not be a charge for re-running a failed sequence if the error is found to have been due to CEQ 8000 instrument error or is otherwise our fault.
Fees for Non-Sequencing Services
Researchers must contact the Laboratory Manger for submission information and to schedule service.
- All Researchers
- *PCR $0.25
- 7- well 1% agarose gel with mass ladder $2
- **USB ExoSAP-IT ® PCR Purification $1.50
- Qiagen QIA quick PCR Purification $2
- Agarase PCR Purification $1.50
- *Researcher must provide optimized protocol and supply DNA template, PCR reagents and primers.
- ** Researcher must supply template in flat-top PCR tube.
Laboratory Hours
The Molecular Core Facility will be open from 9:00 A.M. until 4:00 P.M. Monday through Friday. Please contact the laboratory manager with any questions regarding holiday hours or to request after hours access.
Contact
Tricia Roth
Manager of the Molecular Core Facility
Grice Marine Lab - Room 210
College of Charleston
Phone: (843) 953-9193
Fax: (843) 953-9199
Email: rotht@cofc.edu
Forms
Viewing and Editing Programs
Sequence results will be sent to researchers as SCF files (Standard Chromatogram Files) unless otherwise requested. The programs listed below will allow users to view chromatograms and edit base calls. Please note that in addition to those listed below, other commercial, freeware and shareware programs are available.
Windows
Macintosh
Suggested Vendors
Resources
Links
Policies and pricing last updated: 10 Feb 2008