The GSL only accepts ready-to-load 96-well plates.

Users are responsible for preparation and clean-up of their samples prior to submission of the plate and have access to GSL for these procedures.

We provide training for these steps and then perform the maintenance and loading of the capillary sequencers. Genemapper Analysis software version 4.0 is available at the GSL and Peak Scanner software version 1.0 is 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 multiplexing capabilities.

• http://www.appliedbiosystems.com/
• AFLP User Guide 
• SNPlex User Guide 
• SNPlex Quick Reference Guide 
• 3730xl Dye Card

Genotyping Steps

1. DNA extraction via commercial kits, phenol extraction, etc.
2. Quantitate 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, HiDi Formamide, and size standard
6. Signup on the equipment calendar
7. Submit plate and plate record
8. Data Retrieval

Genotyping Reaction Setup

Denature plate at 95° C for 3-5 minutes. Place on ice. 

PCR Template

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.

Size Standard

LIZ-600 is the available size standard for the dye set optimized on the 3730xl sequencer. 0.5uL per reaction.

HiDi Formamide

Users will need to purchase highly deionized formamide from Applied Biosystems. 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 10µL. Empty wells should be filled with 10 µL of water.

Plate Submission

To submit your plate to the GRL, first turn on the heat sealer and wait for the light to start blinking. Once the heater has reached temperature, place plate in sealer, cover with heat sealing foil (White side up), press and hold for 3-8 sec. See image below. Then, place your clearly labeled plate in the 4°C refrigerator in the box marked “fingerprinting.”

Plate Record Submission

You must submit a plate record before your plate can be sequenced. The plate record must match the name that is physically on your plate. The plate name and sample names must 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:

• Create an excel sheet and list your sample names in 1 single column. The order should go DOWN your plate columns, NOT across. See image below:

• Submit online using the Fingerprint plate submission link on your user menu or the link below:
  

• Fingerprint Plate
  

• Continue Login
• Sign on with UNITY ID and PASSWORD

• Enter plate name and description (optional)
• Copy and Paste the single column from excel
• Submit

Data Retrieval

Each PI will receive a unique URL. Data will be posted on that URL within 1-3 days. The URL will have the following format where “unityid” is the unity id of the PI:

http://service001.hpc.ncsu.edu/GRL/users/unityid/

If necessary, contact Jenn