Dye-terminator sequencing is performed using a modified protocol supplied by Applied Biosystems BigDye Terminator 1.1 and 3.1 Cycle sequencing kits. All reactions are analyzed using a 48-capillary 3730 Genetic Analyzer. Sanger Sequencing can be performed on a variety of templates including PCR product, plasmids, and BACs for analysis of mutation detection and traditional DNA sequencing. Microsatellite Genotyping is also supported by the Core and run on the 3730 Genetic Analyzer.
The Genomics Core offers quality sequencing data, with a fast turn-around time, and knowledgeable help with troubleshooting. Discounts are available for large projects.
Sample Submission
- Samples are processed on Monday and Thursday mornings (9:00 a.m.) with data released the next day.
- Samples and primers MUST be submitted in separate 1.5mL Eppendorf tubes. NO STRIP TUBES.
- Recommended sample volumes and concentrations are listed in the chart below.
- Universal Primers offered are listed in the chart below.
- Clearly label each tube with the appropriate name, the date, and user name/initials. Sample names on tube and on iLab request form should match.
- Large sets of samples can be submitted in a 96-well plate format with a minimum volume of 10ul per reaction. Sample names and well location will be provided by the user in the iLab request.
| Type of Template | Quantity | Volume (uL) |
|---|---|---|
| Plasmid | 100 – 300ng per reaction | 10uL |
| Short PCR product (<500bp) | 50 – 100ng per reaction | 10uL |
| Long PCR product (>500bp) | 100 – 200ng per reaction | 10uL |
| Primer | 20uM concentration | 2uL per reaction |
To view all services offered and pricing, visit our Pricing page.
Universal Primers
The Genomics Core offers universal primers at no cost to the user for plasmid sequencing services. List primer(s) required in the iLab form.
Universal primers stocked by the Core
| Name | Sequence |
|---|---|
| BGH reverse | 5’ TAGAAGGCACAGTCGAGG |
| CMV-for | 5’ CGCAAATGGGCGGTAGGCGTG |
| GLprimer1 | 5’ TGTATCTTATGGTACTGTAACTG |
| GLprimer2 | 5’ CTTTATGTTTTTGGCGTCTTCC |
| M13 Forward | 5’ GTTTTCCCAGTCACGAC |
| M13 Reverse | 5’ AACAGCTATGACCATG |
| pGEX 3′ | CCGGGAGCTGCATGTGTCAGAGG |
| pGEX 5′ | GGGCTGGCAAGCCACGTTTGGTG |
| SP6 | 5’ ATTTAGGTGACACTATA |
| T3 | 5’ AATTACCCTCACTAAAGGG |
| T7 promoter | 5’ TAATACGACTCACTATAGGG |
| T7 terminator | GCTAGTTATTGCTCAGCGGT |
Fragment Analysis
Fragment Analysis, or microsatellite genotyping, is analyzed using the Applied Bio Systems 3730 Genomic Analyzer. Samples should be brought to the Core post-PCR for analysis. Samples should be in plate format with a minimum of 10ul volume. The Core is capable of analyzing dye sets 6-FAM, HEX, NED, ROX, VIC, and PET using a LIZ500-basepair standard or the LIZ600-basepair standard. SNaPshot analysis is also available.
Results
Data is released to users via Google Drive. A folder is created per PI and user and is shared directly with the user. Once a folder share has been accepted, the run files can be downloaded for further analysis. Data will not permanently remain on Google Drive and should be archived by the user, however the Core will have access to backups of sanger sequencing data.