EnGGen provides automated sequencing and fragment
analysis using capillary-based Applied Biosystems 3130 and 3730xl Genetic Analyzers. These instruments offer high throughput and
sensitivity with the ability to handle hundreds of samples per day.
High quality sequences of 800 base pairs or more is common. Fragment
analysis can also be performed for popluar marker methods such as AFLPs, MSATs, tRFLPs and SNPs.
Scheduling your 3130 or 3730xl run:
Current users are expected to be trained on the correct use of the instruments and to sign up for runs ahead of time. Instrument use is on a first-come,
first-served basis. Users may schedule their sequencing or fragment
analysis using webcalendar. Instructions on how to sign up for a 3130
or 3730xl run can be found here. Templates for uploading sample data are available on the resources page.
Preparing your sample:
DNA sequencing reactions should be cleaned, resuspended in 10uL deionized formamide, and denatured (95C 3-5min, 4C 1sec).
PCR products for fragment analysis can be directly added to 10uL formamide containing fluorescent size standard and denatured. Generally, if 2uL PCR products appears "bright" on a gel, 1uL is sufficient for visualization on the capillary. Some calibration by the user may be necessary to achieve the desired signal strength. GS500 LIZ standard (ABI) is a good internal size standard for fragments of 80-500bp. 0.20uL GS500 LIZ per well yields consistent results.
Though the denature cycle and formamide should prevent formation of secondary structures that can compromise your analysis, some users prefer to place their samples on ice for about 5 minutes following denaturation.