Genotyping Service Details
Assay Design Workflow
We will first have a meeting to discuss your research goals and the suitability of a TaqMan-based assay for your region of interest. If the assay design and chemistries are applicable, we’ll need you to provide annotated sequences for your region of interest, including marking the exact site of insertion of your transgene and other genetic variations. From that sequence, we’ll use software to design PCR and TaqMan hybridization probes—one specific for the “wild-type” sequence, the other specific for the insertion or “mutant” allele. Using ~ 20 – 30 samples of known genotype from your lab, we’ll optimize the assay and communicate with you the results of initial optimization. Upon successful validation with a larger dataset (~100-200 samples), and creation of appropriate control samples, you’ll be able to request the assay for genotyping in the LMT Genotyping LIMS.
Sample Processing Workflow
Once mouse-tail lysates are received, LMT staff will record the receipt of your lysate plate in the LMT Genotyping LIMS. A unique numerical identifier is assigned to the 96-well lysate plate, and a “Dilution Plate” is created on a Biomek FX Automation Workstation. Using automation, customer samples will then be setup with corresponding assays in a 384-well plate in duplicate. LMT staff will follow assay-setup instructions (e.g., appropriate Plate ID, assay ID) given in the LIMS. After PCR amplification and end-point fluorescence detection, your genotypes are scored using ABI’s analysis software, which displays fluorescence data in a X-Y plot format for easy clustering and scoring. Results are imported into the LIMS, and any discordance between replicates is automatically flagged and raw data is reviewed by LMT staff. Upon final review of data, an automatic email notification is sent to you stating that an Excel file—containing your genotypes—is available for download from the LIMS.