Fragment Genotyping Guidelines
The GSL only accepts ready-to-load 96-well plates, submitted through our online system.
All plates submitted must be 96-well semi skirted PCR Plates (available for purchase at the GSL). One of the following plate types is required for plate submission:
- USA Scientific: Part Number 1402-9200
- Genesee Scientific: Part Number 27-408
Users are responsible for preparation and clean-up of their samples prior to submission of the plate. We perform the loading of the capillary sequencers.
All Genotyping plates are processed on an Applied Biosystems 3730xl DNA Analyzer. Genotyping on the 3730xl has a wide range of applications including AFLP, Microsatellite, SNP analysis, etc. The 3730xl is optimized for a 5-dye set (Dye set 33 Filter set G5). The dyes in this set include 6-FAM, VIC, NED, PET, and LIZ size standard. The 5-dye set has increased the multiplexing capabilities.
- DNA extraction via commercial kits, phenol extraction etc.
- Quantify DNA using agarose electrophoresis, spectrophotometry, etc.
- Perform PCR with dye labeled primers
- Multiplex by combining PCR products
- Set-up plate with PCR products, Hi-Di Formamide, and size standard
- Submit plate and plate record
- Data retrieval
Genotyping Reaction Setup
- PCR Template: Optimized by user
- Size Standard: 0.5 uL per reaction
- High-Di Formamide: Amount needed to bring final reaction volume to 10 uL.
Users are required to optimize the amount of template best for their samples. If the user desires to multiplex, it is best to run the PCR reactions separately and then combine them in the final plate. You can have up to 4 different PCR products in one well; one for each of the available dyes.
LIZ-600 is the available size standard for the dye set optimized on the 3730xl sequencer. Use 0.5 uL per reaction.
Users will need to purchase highly de-ionized formamide from Life Technologies. It is important to get this grade of formamide because others will affect the sequencing run. Use the formamide to bring the final volume of the reaction to 10uL. Empty wells should be filled with 10uL of water.
Plate Record Submission
You must attach a plate record to your online submission (templates are available in the submission portal). The plate record must match the name that is physically on your plate. The plate name and sample names should follow the rules below:
- Must be 10 characters or less
- No spaces
- No unusual characters; you may use underscores __
- Name all wells, if needed use blank, empty, water, etc.
To make a plate record
- Download the plate submission sheet (through the submission portal) and list your sample names in 1 single column. The order should go DOWN your plate columns, NOT across.
- Save the plate record as a tab delineated text file (.txt)
- Upload the file to your request in the submission system.
Data will be emailed from the GSLNCSU@gmail.com account.