Genotyping Guidelines

Genotyping Services

The LBB core lab prefers genotyping samples to come in ready-to-load 96-well plates, however if you need assistance setting up your plates we help you out. 

All plates submitted must be 96-well semi skirted PCR Plates. 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.  

LBB provides assistance and direction for sample setup and then perform the maintenance and loading of the capillary sequencers. The data in .fsa format can be viewed using Genemapper Analysis software version 5.0 and is available at LBB. Other software such as Peak Scanner Software (version 1. 0) is also  available online.  

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 and allows for up to 4 different dye labels to be multiplexed with the same sample.  

 
Genotyping Steps

  1. DNA extraction via commercial kits, phenol extraction etc.
  2. Quantify DNA using agarose electrophoresis, spectrophotometry, etc.
  3. Perform PCR with dye labeled primers
  4. Multiplex by combining PCR products
  5. Set-up plate with PCR products, Hi-Di Formamide, and size standard
  6. Submit plate and plate record
  7. 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.

 

PCR Template

Users are required to optimize the amount of template best for their samples and a dilution series is recommended for first time users.  If the user desires to multiplex, it is best to run the PCR reactions separately and then combine them in the final plate. It is possible to multiplex at PCR, however individual optimization is critical.  You can have up to 4 different PCR products in one well; one for each of the available dyes.

 
Size Standard

LIZ-500 is the available size standard at LBB for the dye set optimized on the 3730xl sequencer.  We recommend 0.5 uL per reaction, however users often dilute with adequate results. Other size standards such as Liz-600 and Liz-1200 are also available for purchase separately.  

 
Hi-Di Formamide

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 HiDi. 

 
Plate Record Submission

You must submit a sample sheet at the time of sample submission. Sample sheets are available at the core or can also be e-mailed upon request. We find that keeping the sample names as simple as possible is best. Less than 10 characters is good, and there can be no spaces or non-standard characters (- or _ are ok)  in the name. 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.

 

Data Retrieval

WSU users will have access to the Office of Research Data server at this location : \\lbbfileserver.or.wsu.edu\pi_folders 

Please contact Derek Pouchnik (pooch@wsu.edu) to request access to your PI folder.