Services Offered
| Automated DNA Sequencing | [Back to the top of this page] |
The DNA sequencing core is used by researchers for sequencing of plasmids, PCR products and large insert clones for gene discovery as well as for mutation detection and diagnostics. Optimal results are achieved on clean, purified templates and well-designed primers. Each sequencing run (one template/one primer) typically produces 600-800 nucleotides of high-quality sequence data. The Biomolecular Core Laboratory offers a full range of sequencing services.
Sample Run Only Sequencing Services
The users perform cycle sequencing reactions using fluorescent dye terminators based on either ABI or Amersham chemistry, and provide the cleaned up reactions in 0.20 ml tubes to the core facility along with a 3130 run only submission form. The BCL staffs process the samples for capillary electrophoresis on an ABI 3130 Genetic Analyzer. After data collection and processing, customers receive a sequence text file and chromatogram. Turnaround time for basic sequencing is 24 to 48 hrs.
Full Sequencing Services
The Core also provides full service sequencing by advance request only. Template DNA (pure plasmids, PCR products, and gel-isolated fragments) and primers are provided by the user and the core processes them using a BigDye Terminator Cycle sequencing kit. Customers must submit the Full sequencing request form along with the gel picture of the DNA template at the time of submission.
| DNA Fragment Analysis | [Back to the top of this page] |
The DNA Fragment Analysis Core is currently used for quantitative analysis, mutation detection, and genotyping for linkage analysis. Fluorescent-tagged DNA fragments are separated by electrophoresis and analyzed using either Genescan with Genotyper or GeneMapper software. Several levels of services are offered depending on the needs of the researchers. We also offer Genotyping software training to anyone interested. For inquiries on project design and development, individual researchers should consult with the Biomolecular Core Director, Katia Sol-Church, PhD. (Please see the Contact Us page for contact information.)
Fragment Analysis on the ABI 310
The following dye sets can be run on the 310:
- 6-FAM, TET, HEX, with TAMRA standard (virtual filter set C)
- 6-FAM, HEX or VIC, NED, with ROX standard (virtual filter set D)
- 5-FAM, JOE, NED, with ROX standard (virtual filter set F)
Dyes from one filter set cannot be analyzed at the same time as dyes from the other set, so don't "mix 'n' match" dyes.
Please contact the Core the day before you would like your samples to be run. Each samples takes 30 minutes to run. If the instrument is full for that day, your samples will be placed in a queue for overnight or next day processing. Samples are typically run overnight with results ready the next business day.
Fragment Analysis on the ABI 3130
The following dye sets can be run on the 3130:
- 6-FAM, VIC, NED, PET with LIZ standard (from the G5 set)
- 6-FAM, HEX, NED, ROX (from Filter set D)
- 5-FAM, JOE, NED with ROX standard (from Filter set F)
Dyes from one filter set cannot be analyzed at the same time as dyes from the other set, so don't "mix 'n' match" dyes.
Please contact the Core the day before you would like your samples to be run. For capillary electrophoresis on the ABI 3130xl, 16 samples are run at the same time on a 16-capillary array. Advance notification is required to ensure availability of the machine.
Long Fragment Analysis
For fragment analysis of products up to 1200 base pairs in length, the following dye set can be run on the 3130:
- 6-FAM, VIC, NED, PET with LIZ standard (from the G5 set)
| Real-Time PCR | [Back to the top of this page] |
The Core is home to an ABI 7900HT Fast Real-Time PCR system. It contains a 384 well plate block. After instrument training, investigators can set up a Real-Time PCR plate and run the instrument. Data is stored on the Core computer, but can transferred after the run to either a flash, CD or other external media. SyberGreen, TaqMan, Eclipse, Scorpio and SuperArray technologies are routinely used on this instrument.
| Pyrosequencing | [Back to the top of this page] |
Currently used by investigators for SNP/mutation detection analysis, allelic frequency, haplotyping and short-read sequence analysis. Please contact the Core lab for additional Pyrosequencing information including a more detailed protocol packet, which is available upon request.
Following the manufacturer's guidelines, the researcher will set up and perform the initial PCR reaction. The Core will provide the vacuum tool, which must be used in Room 214 of Rockland Center One. Please sign up for the machine and coordinate the use of the vacuum tool and lab bench space to set-up the pyrosequencing reaction at least one day in advance.
| Bioanalyzer | [Back to the top of this page] |
Used by investigators to determine RNA quality or DNA sizing. The Core lab has in stock the Agilent DNA 1000 kit and the Agilent RNA 6000 Nano kit for customer use. Other kits are also available. Please contact Agilent for a complete list of Bioanalyzer reagents. The Core lab has information (including technical guides and protocols) on some Bioanalyzer reagent kits. We have the ability to run the RNA, DNA and Protein kits, but not the cell solutions kits.
| Robotic Stations | [Back to the top of this page] |
The Biomolecular Core Laboratory houses a Stratagene Robocycler and a Biomek 2000 liquid handling workstation.
Robocycler
The Robocycler PCR machine has three separate heat blocks one of which has a gradient capability. This gradient block can be used to determine the optimal temperature conditions for difficult templates. In addition, the separate heat blocks mean no sample ramping time thus reducing overall run time. A sign up sheet is posted next to the thermocycler to avoid congestion. Complimentary training sessions are available upon request.
Biomek 2000
Designed for high throughput automatic pipetting, the Biomek 2000 is routinely used to set up multiplex PCR reactions in 96 well format or strip tubes, pooling PCR reactions, and diluting DNA samples prior to gel electrophoresis. It is also routinely used for aliquoting reagents used in the setup of 384 well Real-Time PCR plates.
Tools for the Biomek include a single channel P200L (with level sensing feature), a multichannel MP20 (20ul) and a multichannel MP200 (200ul, 8 channels). BCL staff will provide consultation for experimental set up and training as needed.
| Custom Oligonucleotides | [Back to the top of this page] |
BCL has a purchasing agreement with Integrated DNA Technologies (IDT) to obtain high quality oligonucleotides at reduced cost. The turn around time for standard primer is two days. Modifications such as fluorescent labeling/HPLC purification or dual (Real-Time) probes require 5-7 days for synthesis. Oligo's can be picked up at the core facility during business hours (M-F 9:00 AM - 4:00 PM).
For general IDT information please visit: www.idtdna.com
To access Nemours oligo ordering portal go to: www.idtdna.com/NEMOURW-BioMed
Mitch Gore, IDT sales consultant. Phone:610-524-1350
Please let us know via the Contact Us page if you would like his email address.
| Support for Diagnostic Testing Laboratories | [Back to the top of this page] |
Operating under strict CLIA guidelines, BCL sequencing services currently supports laboratories offering diagnostic tests for the following diseases:
- Barth Syndrome
- Benign Hereditory Chorea
- Costello Syndrome
- Cranio-Facial-Cardio Syndrome
- Duchenne Muscular Dystrophy
- Fiengold Syndrome
- Pelizaeus-Merzbacher Disease
- Pelizeaus-Merzbacher-Like Disease
- Rett syndrome
- Spinal Muscular Atrophy
| Consultations | [Back to the top of this page] |
Consultations on project design for hypothesis-based research and for the development of new molecular diagnostics tools are ongoing. Quotes are available upon request for project's budget developments. Interested individuals should set up an appointment by contacting the laboratory director, Dr. Katia Sol-Church via phone (302-651-6705) or email (using the Contact Us page).