General Information

Single tube samples - please use the Genewiz drop box - details here.

The GSL performs all sequencing reactions on an Applied Biosystems 3730xl DNA Analyzer. For more information about our equipment, please follow the Instrumentation link. We currently have three different levels of sequencing service available: Platinum, Gold and Bronze. We can provide training for all of these steps and then perform the maintenance and loading of the capillary sequencers. Sequencing Analysis software version 5.2 is available at the GSL. Other analysis software is available online such as Sequencing Scanner Software V1.0 and Vector NTI.

• http://www.appliedbiosystems.com/
• Automated DNA Sequencing Chemistry Guide
• 3730xl Sequencing Chemistry Guide

PCR Clean-up Service

 For PCR clean-up, samples MUST be submitted in 96-well, semi-skirted PCR plates with a reaction volume of 10μL.

Please contact Jenn or Megan for scheduling this service.  

 This level of service includes the following:

Sequencing Reaction set-up

Sequencing Reaction clean-up

Sequencing Run and Data Distribution

When sequencing at the Gold level, please submit your samples in a 96 well plate at 2X DNA template quantity (see table) with 6.4pmols of primer (either forward or reverse).The final volume should be 12μl. Follow the standard protocol for plate submission, plate record submission and data retrieval described below. Please label your plate with the exact same name as the plate record you submit online. Make sure that you sign the log book next to the heat sealer in our lab.

2X Template Quantity used for Gold Sequencing:

When sequencing at the bronze level, users are responsible for the preparation and clean-up of their samples prior to submission of the plate. Training can be provided and users have access to the GSL for these procedures. Applied Biosystems Big Dye 3.1 must be purchased at the GSL and is required for all sequencing reactions. This mixture contains the Taq, 2.5X buffer, Mg²⁺, dNTPs and dye-labeled ddNTPs. Simply add your primer (forward or reverse). A clean-up plate must also be purchased at the GSL (included in service price) before it is processed on the DNA analyzer. Information about the reaction set-up and clean-up protocol appears below.

Sequencing Steps

1. Template (single-stranded, double stranded, plasmid, BAC, PCR products) preparation via commercial kits, phenol extraction, plasmid prep, etc.
2. Purification of product- removal of primers, dNTPs, and other contaminants with ethanol precipitation, commercial kits, etc.
3. Quantitate DNA using agarose electrophoresis, spectrometry, etc.
4. Amplification via cycle sequencing with Big Dye (purchase at GSL), primer, and template
5. Edge clean-up of samples (purchase at GSL)
6. Submit plate and plate record
7. Data Retrieval

Sequencing Reaction Setup (BRONZE)

Template Quantitation

Applied Biosystems Recommended Sequencing Template Concentrations

Full reaction per sample

1/2 reaction per sample

1/4 reaction per sample

Cycle Sequencing

For Single and Double Stranded DNA

For BAC DNA 

Plate Submission

Please label the plate with 1) the name matching your plate record 2) the level of sequencing service you are requesting (Gold or Bronze)

Place Gold sequencing plates (samples that still need the sequencing reaction set-up) in the 4 degree cooler in the box labeled "Drop Off."

Place Bronze sequencing plates (samples that have been through the sequencing reaction but need to be cleaned) in the 4 degree cooler in the box labeled "To Be Clean-Seq"

Sign the billing form and sequencing log.

**Remember** Plates must be 96-well, semi-skirted

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 plate submission link on your user menu or the appropriate link below:
  

• Regular Plate (if all samples have different names)
• Blue Plate (if all samples have the same name) 

• 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/GSL/users/unityid/