Fragment analysis (Genotyping) can be performed on DNA fragments that have fluorescent labels. Using a labeled primer with PCR amplification is a common method used to incorporate these labels. The Molecular Biology Core lab is already set to run multiple fluorescent dye sets.
6-FAM, VIC, NED, PET, LIZ (for ABI Dye Set G5, DS-33)
6-FAM, HEX, NED, ROX (for ABI Dye Set D, DS-30)
Post PCR reaction products should be visible on a gel as a strong band. For capillary electrophoresis analysis samples need to be diluted down to a working concentration. Our 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 0.25ul of your selected size standard (we recommend ABI GS500 LIZ ABI# 4322682 or GS500 ROX ABI #401734 for the Dye set listed above) for a total reaction volume of 10.25ul for each sample.
The amount of the PCR 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 or similar plate) before you give them to the core. 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 (column 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. See Instructions for submitting samples in 96 well plates for more information.
- 20ul gDNA @ 100ng/ul
- Unpurified Tagged /labeled PCR product
- purified Tagged /labeled PCR product
- Provide the entire samples name according to the plate format. If you need sizing information please provide the details of labeling and product size. Please find the order form here (link)
- Compiled report
- .FSA file