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DNA Sequencing

The Genomics Core Sequencing Laboratory utilizes the 3130xl automated, 16-capillary DNA sequencer. Applied Biosystems BigDye terminator cycle-sequencing kits are optimized dideoxy chain-termination reactions employed to flourescently label DNA fragments. Products are eletrophoresed through the 36cm array and POP-7 polymer is used as the separation matrix before detection by the charge-coupled device (CCD) camera. This machine is equipped with two autosamplers, making it possible to acquire data for over 700 samples per day, with an average read length of 500 bases each.

Example applications include confirming vector recombinantion, gene mutation and SNP detection, genotyping, and diagnostic assay development.

We offer basic and full-service packages:

Full service

Customer provides: Purified template DNA and sequencing primer (see preparation requirements below)

Genomics Core Facility Provides:

• Amplification of DNA template with ABI BigDye Terminator Cycle Sequencing kit. Every cycle sequencing reaction set includes a PCR control reaction for each type of chemistry (v1.1 or v3.1) used (pGem-3Zf+, -21M13F primer)
• Sephadex purification and vacuum drying of extension products
• Re-suspension of dried sample in HiDi formamide
• Denaturation
• Data collection software setup
• Sample electrophoresis on 3130xl Genetic Analyzer that includes manufacturer provided sequencing standards as array controls for each type of chemistry used
• Sequence analysis/base calling software setup
• Data transfer

Typically, full-service sequencing expected turnaround time is one business day.

Basic service

Customer performs: Cycle sequence reactions and purification of extension products. (We strongly recommend including a PCR control reaction for each type of chemistry used.)

Genomics Core Facility Provides:

• Vacuum drying of extension products
• Re-suspension of pure, dried extension products in HiDi formamide
• Denaturation
• Data collection software setup
• Sample electrophoresis on 3130xl Genetic Analyzer that includes manufacturer provided sequencing standards as array controls for each type of chemistry used
• Sequence analysis/base calling software setup
• Data transfer

Basic service samples submitted by 10a.m. will be sequenced by 5 p.m. on the same day.

Sequencing Template Preparation, Quantity, and Quality Requirements

Results

Sequence Analysis v5.2 software is employed to provide preliminary analysis of sequence data. This includes KBTM Basecaller, visual examination of raw data and electropherograms by a trained technician, and a sequence quality report (QV and LOR scores for each sequencing reaction). If quality control sequences (Both pGEMZf+ and standards) meet acceptable limits, the Genomics Core Facility is not responsible for re-sequencing failed samples. However, we will be more than happy to provide consultation and assist wtih troubleshooting. For investigators performing their own cycle sequencing, we strongly recommend including a PCR control reaction for each type of chemistry used.

Data Transfer

When submitting samples, investigators need to specify data transfer preferences. Sequence files (*.ab1, *.phd) can be sent via e-mail, stored, and sent on DVD and/or hard copies (printouts). Sequence data can be sent via Postal Service.

All data will be stored in our database for one month, and will be available for re-sending up until that point.

Check out our Useful Links for downloadable freeware that can be used to view your sequence traces.