FAQs - Fragment Analysis
IMPORTANT NOTE
Data output files will require specialized software in order to be viewed. Those who do not have access to Genemapper or Genotyper from Applied Biosystems may inquire about using the site licensed copy installed at the Biotechnology Computing Facility. There are also a few freeware viewers with varying levels of capability.
Sample Preparation
PCR should be performed by the investigator using dye labeled primers. Reactions do not require special preparation before using the GATC facility. Multiple markers may be run in the same lane if they are separated either by size or by the fluorescent label color.
Multiplex PCR is the most labor efficient means of maximizing information gained per injection. However, the more primer pairs one attempts to involve in one reaction, the higher the likelihood of detrimental primer-primer interaction. Multiplex PCR will require some optimization and therefore is best employed when studying large populations.
For smaller studies, PCR may be performed one marker at a time then mixed post-PCR. If samples are to be multiplexed post-PCR, the user should mix the fragments in the appropriate ratios before submitting. Keep in mind that for each additional marker added, all markers will be effectively diluted to 1/n of their original signal strength, where n is the number of markers added.
Several dye sets are supported on our ABI 3730. The dye sets are discrete, so dyes from one set cannot be combined with another. The following dye sets may be used:
Dye set D: 6-FAM (Blue), HEX (Green), NED (Yellow).
Dye set G5: 6-FAM, VIC (Green), NED, PET (Red).
Note: 6-FAM and HEX are commonly available through vendors such as Invitrogen, Operon, and others. VIC, NED, and PET are available from Applied Biosystems. The additional color in the G5 dye set allows for a higher degree of multiplexing.
What We Do
Prior to capillary electrophoresis, the PCR is mixed with a sizing standard (GS500 from Applied Biosystems p/n: 401734). The standard is used to create a regression equation which is then used to calculate the sizes of unknown fragments in the lane. GS 500 standard is provided by the GATC, and is mixed using the following ratio: 0.003ul Ladder, 9ul Hi-Di Formamide (Applied Biosystems p/n: 4311320), 1ul PCR product (diluted to optimal concentration- usually 1:20 with H 2 O).
This mixture is denatured at 95 ° C for 5 minutes then cooled to 4 ° C. Then the plate is loaded into the 3730 DNA Analyzer. Electrophoresis is performed with a 36cm capillary using the default microsatellite module and POP-7 polymer.
