Sanger Sequencing Service Details
Sample Processing Workflow
Once PCR products, plasmids, or BACs are received, LMT staff will purify the template either via enzyme (Exonuclease and Shrimp Alkaline Phosphatase) or filtration (Sephadex® G50 on MultiScreen Plate). An aliquot of purified template will be used for the sequencing reaction, using Invitrogen’s BigDye® Terminator chemistry, and either commonly used primers provided by LMT, or specific sequencing primers sent by you. The sequencing reaction will consume less than half of the purified template, which will allow for the sample to be repeated in the event of poor quality data and per the customer’s request. The sequencing products are then purified using magnetic beads (Agencourt’s CleanSeq®), and purified products are loaded onto the ABI 3730xl sequencing instrument.
Data Analysis Workflow
After electrophoresis, chromatograms and fasta files (containing text sequence) are automatically sent from the ABI 3730xl to the Sanger Sequencing LIMS. Sequence data is reviewed by lab staff, reviewing control data and trimming poor quality sequence where necessary, and staff assigns a status of “approved” or “reject” to each sample. After review, automated e-mails are sent to you notifying that both chromatogram and fasta files are available for you to download from your secure folder in the LIMS. Typically, data files are available 24-48 hours after sample receipt. Any suboptimal results are followed up by a personal phone call or e-mail from our staff to decide further steps.