Capillary-Based Sequencing Sample Prep
Detailed protocols on template preparation, PCR reaction mix preparation, cycling conditions, and post-PCR purification are available at http://www.appliedbiosystems.com. The protocols are summarized below.
We recommend the Qiagen miniprep kit for isolating plasmid DNA and the Qiagen PCR purification kit to prepare PCR products.
Setting up Sequencing PCR reaction
The standard reaction can be halved or quartered to economize the BigDye Mix.
*BigDye Reaction Mix contains 2.5X Sequencing Buffer. This must be supplemented with 5X Sequencing Buffer in quarter volume reactions.
PCR Cycle Sequencing
25 cycles of
store at -20°C until ready to purify the reaction samples.
An MJ Research (Biorad) Tetrad thermocylcer programmed with these cycling conditions is available in the Bauer Core. It is saved in the Main menu and called “BIGDYE”.
We strongly recommend column purification of the PCR sequencing samples. We most commonly use Centrisep columns from Princeton Separation, but similar products work as well. Samples may be resuspended in water or formamide at a minimum volume of 10uL. Samples must be submitted in 8-strip tubes (0.2uL tubes) or plates.
Following is a list of reagents mentioned in this document.
The Sequencing Facility sells 100-reaction aliquots of BigDye Terminator Reaction Mix. To purchase an aliquot, please fill out this form and email it to email@example.com. You can pick up your aliquot from the sequencing staff in NWL room B239. 1mL aliquots of 5X Sequencing Buffer are available free of charge to BigDye customers.