The GLGC has an ABI 3730 DNA Analyzer that runs a 50cm 48 capillary array and is used for both Sanger sequencing and fragment analysis. POP7 polymer is used exclusively on this instrument, and BigDye 3.1 is used for all sequencing reactions. Our 3730 routinely provides up to 800 bases of usable sequence per reaction, and we are capable of sequencing short (>300 base pair) PCR amplicons. We can also process microsatellites, AFLPs, and SNPs.
We provide 2 levels of service: full service sequencing or microsatellite genotyping and ready to load plates.
Full Service Sequencing
User must provide quantified DNA template and custom primer. We do provide some commonly used plasmid primers free of charge.
We will set up, cycle, clean, and analyze the reaction.
You must provide DNA and primers at the following concentrations and volumes:
|Please provide the following:|
|Plasmid DNA||100-200 ng/μl||10 /μl reaction|
|PCR fragment||3-8 ng/μl||10 /μl reaction|
|Primer||10-20 pmole||10 /μl reaction|
*A sequencing reaction only uses 1 primer per reaction. If you would like to sequence in the opposite direction another reaction needs to be set-up with the other primer.
*Primers must be removed from PCR fragments before sequencing.
Full Service Microsatellite Genotyping
User must provide DNA and fluorescently tagged primers. We will set up, cycle, and analyze the reaction. Reaction set up information and thermocycler profile needs to be provided by the user. Optimization of PCR reactions is not included in this service and must be done by the user.
The 3730 is calibrated for the DS30 4 color dye set and G5 DS33 5 color dye set. We can calibrate to other ThermoFisher/Invitrogen dye sets if needed.
Ready to Load Plates – Sequencing and Fragment Analysis
The user runs their own reactions, removes primers and unincorporated nucleotides, and resuspends the samples in at least 10 µL of formamide. Wells that do not contain a sample must be filled with 10 µL of water or formamide. Be sure to use a half-skirted plate compatible with an ABI 3730 DNA Analyzer. We recommend these: Dot Scientific, catalog #951-PCR.
Training is available from the GLGC if you would like to set-up your own sequencing reactions and prepare ready to load plates.
If you are unsure of what type of service you require, contact us for a consultation and quote.
We will provide both .ab1 and .seq files of sequencing data for each sample.
We will provide a .fsa file for each fragment analysis sample.
The GLGC has available for use CLC-Genomics Workbench and DNAStar for sequencing data analysis and GeneMapper for .fsa files.
The Great Lakes Genomics Center strives to provide the best data possible in a timely manner. Turn around time is typically 3 days for full service sequencing and fragment analysis and 1 day for ready to load plates.
In the advent of failed services, GLGC staff will work with the researcher to identify the cause. If sample quality or researcher error is identified as the cause, the user will be responsible for the full contractual agreement. If the cause is due to equipment failure or GLGC staff error, the service will be repeated without additional cost to the user. GLGC is never financially responsible for the cost associated with sample obtainment (ie DNA prep, RNA prep, experiment costs, etc.).