Automated Fluorescent DNA Fragment Analaysis in the CGC
3730 and 3130xl Genetic Analyzers can be used to accurately determine the size of fluorescently labelled PCR products and other types of fluorescently labelled DNA fragments in addition to DNA Sequencing.
Fragment analysis experiments will generally use a short capillary array suitable for use with 400bp or 500bp ROX- or LIZ- labelled size standard ladders. The 3730 in the CGC is configured with a 50cm array and calibrated for the DS30 4 color dye set, which uses ROX as the size standard label. It can also be calibrated for the 5 color G5 dye set on request. The 50cm array is longer than the one typically used for fragment analysis (36cm), but use of this array allows greater flexibility as it allows switching between sequencing runs and fragment analysis runs without swapping arrays, at the cost of somewhat longer run times. The throughput afforded by the 3 fold larger number of capillaries compared to our previous instrument (ABI 3100) more than offsets the extra run time.
Users are expected to provide their own consumable supplies, such as their PCR mixes and primers, 96-well plates and sealing film, and fluorescent size standards.
What you can buy from the CGC:
- Flat Top flexible 96-well plates. These can be used for PCR reactions, cycle sequence reactions, and for loading samples into the 3130xl or 3730.
- Plate sealing film, for running PCR and cycle sequencing reactions.
- ABI plate sealing septa, for loading plates in the 3130xl or 3730.
- 400HD ROX size standard.
See the CGC Store list for the details.
The standard configuration for the ABI 3730, a 50cm array running POP7 polymer, allows either fragment analysis or sequence runs to be made in one hour for $48. Projects that use a 1000bp size standard ladder can run samples on the 3130XL. This type of run is roughly equivalent to a long read sequencing run and takes about 3 hours, for $21.
Before each run fresh polymer is pumped into all of the capillaries in the array regardless of the actual number of samples being analyzed. Therefore, it is wise to plan runs in groups of 48/16 samples for best cost efficiency. The data collection software allows users to define analysis methods for each run (of 48/16 samples) loaded in a plate, which makes sharing plates with other users possible.
User's accounts will be charged $21.00 per run for either long fragment analysis or long sequencing runs on the 3130XL, or $48.00 for runs on the 3730. Use of the instrument will always be charged on a per-run basis because that is the basis on which costs are actually incurred. The ABI sign out sheets include the instruction that users sharing a run can divide the charge to different budget numbers in sixteenths or 48's of a run increments, but that any empty wells in a run will be divided equally between the users of the run.
Fragment Analysis Service
Plates of fragment analysis samples to be run on the 3730 can be submitted by O/N or ground shipment from customers in North America. The submitter should have created any multiplex mixtures and created a table of sample names to be used in the run. The samples can be sent dried in the plate, or on gel-ice or dry ice as aqueous PCR products mixed as desired and we will add ROX size standard and HiDi formamide, or you can send PCR products in HiDi formamide with ROX or LIZ size standard already mixed. 1-4 96-well plates (or 1 384-well plate) of samples can be processed for $2.00 per sample, 5-8 plates (or two 384-well plates) for $1.85 per sample, or 9+ plates (3 or more 384-well plates) for $1.75 per sample. Add ten cents per sample if we add the size standard and HiDi. You can expect turn-around times of 1-3 business days for the data. Data files will be returned via DropBox link. Call the CGC Director at 206-616-0216 to discuss shipping and sample particulars and any special sample/plate/project naming schemes you would like to have used to streamline the data organization and analysis for your project. Also see the external rates summary for information about how invoices are prepared.
The ABI instruments come with a software package which allows the computer to operate the machine and collect raw data files from it, the DataCollection 3.0 package. For fragment analysis you will be using GeneMapper 3.7 or 4.0 as the data analysis application to organize and analyze the data produced. GeneMapper is a fairly complex application with a significant learning curve. The Center has training CD's which walk the user through the program and allow the user to practice as much as they want as the "course" progresses. Taking the time to work with the training CD is highly recommended for anyone planning to do fragment analysis experiments.
When you are planning an experiment it is worth checking the fragment analysis section of the protocols archive page to see if another user may have found a better or less expensive way to do the kind of experiment you have in mind. The center posts useful protocols and user tips on that page when a user finds a method or experimental design they think is especially good. The protocol archives page also contains a listing of the fluorescent dyes which can be used for labelling primers, and the "filter sets" used by the ABI Genetic Analyzers to collect and analyze data from fragment analysis experiments. You can refer to this information when designing primers.