Introduction | Sample preparation | Submission process | Troubleshooting
Introduction
This service begins with the removal of unincorporated dye terminators from completed sequencing reactions
using an EDTA/ethanol precipitation method.
Samples are then injected in a capillary electrophoresis DNA Analyzer for sequence generation.
Currently, only sequencing reactions performed using the ABI BigDye v1.1 are supported.
Sample preparation
Submit your completed sequencing reactions in 8-tube strips or 96-well plates. The volume of sample must be at least 15ul.
If you are running reactions smaller than 15ul, expand them to 15ul with molecular biology grade water before submitting.
Our automated protocol requires the use of plasticware (tubes, strips & 96-well plates) of uniform dimensions that we provide at no charge. You can find the plasticware at any of our drop-off locations near the barcoding station.
The use of plasticware with improper dimension will delay the processing of your samples.
For this particular service, the use of low-profile 8-tube strips for the fast thermocyclers from Applied Biosystems is allowed but the plasticware is not provided by the facility. To help ensure the proper identification of the orientation of samples within an 8-strip vial, we ask that these strips not be clipped down to fewer than the 8 original wells present in the vial.
Submission process
• Use the username and password we provided you when you registered your account and log in to the system from any page of the website.
• Select the online submission form and choose the SEQUENCING submission type.
• The prompts will guide you through the process. You will need to have the following information to fill out the electronic form: name of submission, type of plasticware in which the samples will be submitted (strips, plates), names of samples and how many you are submitting.
• Make sure to hit the ACCEPT button at the end of the process to complete your submission.
• You will receive an email with an invoice as a PDF attachment for your records.
• Proceed to the delivery of samples to one of our drop-off locations (CCHMC or GRI) and register them using the barcode scanner as indicated in the how to submit section.
Troubleshooting
-use of incompatible chemistry
-too little template
-too much template or primer
-no priming site
-structure in the sequence or high GC content
-multiple templates
-incomplete cleanup