DNA Fragment Analysis
Fragment analysis (Genotyping) can be preformed on DNA fragments that have fluorescent labels. Using a labeled primer with PCR amplification is a common method used to incorporate these labels. The Core lab is already set to run multiple fluorescent labels in several possible combinations.
5-FAM, JOE, NED, with a ROX standard (for ABI Dye Set F)
6-FAM, HEX, NED, with a ROX standard (for ABI Dye Set D)
Other fluorescent labels and combinations are possible. Please contact the Core Facility for more information.
Post PCR reaction mircosatellites should be visible on a gel as a strong band. For capillary electrophoresis analysis samples need to be diluted down to a working concentration. A recommended starting point would be about 1 in 50 dilution of the PCR product. Add 1ul of this dilution to 10ul of Hi-Di Formamide (ABI# 4311320), along with 0.5ul of your selected size standard (we recommend ABI GS500 ROX #401734 for the Dye set listed above) and 8.5ml of Milli-Q water for a total reaction volume of 20ul The amount of the mircosatellite product used for each reaction may need to be further optimized. With too much product the signals can be saturated preventing the software from reading some of the fragments or the size standard signals. Too little product and fragments may not be detected above the background. Adjustments will need to be made based on the amount PCR product generated. Multiple amplicons generated in a single reaction may need to be more dilute while a single amplicon may need to be less dilute.
The reactions need to be set up in an approved 96 well plate (ABI cat# N8010560) before you give them to the core facility. Samples are run by the sequencer by every other column on the plate, so set up samples by filling all the odd number wells first (columns 1A through H followed 3,5,7, 9 and 11 A through H. Then fill all the even numbered columns 2,4,6,8,10, 12 A through H.
Samples are processed 48 at a time for each run, which is about an 1 hours long. Once all the samples are complete the raw data is posted on the MB Core Server for you to retreve by using your MB Core account. For analysis and viewing of the data we recommend the “Peak Scanner” software, which is free software that can be down loaded from the ABI web site. Peak Scanner identifies peaks and fragment sizes up to 1200bp in size from ABI capillary electrophoresis assays. The software allows you to annotate data with labeling, simultaneous viewing of raw and analyzed data, and allows the organization of the sample files into projects. Other software options are available but most are not free. For more information contact the Core Facility.