in Chet Fornari's Lab with Dr. Paula Evans, Biochemist and Sequencing Lab Supervisor
Olin Building - Sequencing Lab - room 219
Lab funded by:
(1) template preparations
(2) cycle-sequencing rxns
(3) product purification & preparations for the CEQ 8000
(4) sequencing runs in the CEQ
(5) data export to lab network I:/drives
(6) bioinformatic analyses of collected data
We have developed (1) template preparation methods for both plasmid vectors and PCR fragments, (2) cycle-sequencing reactions, and (3) reaction product purification methods for both plasmid and PCR-fragment templates for use with Beckman-Coulter's DTCS kit and the CEQ 8000 Automated Gene Analysis instrument. Please take a look at the side-bar for the 6 parts of a typical sequencing project.
Our protocols are based primarily on methods and techniques provided by Beckman-Coulter in its sequencing manual but we have modified some procedures and added some new ones. These modified protocols work very well for almost any sequencing project (> 700 base calls of high qualities and ~ 900 with full reactions). We typically use less than a full reaction, which has 8 ul DTCS Master Mix in a 20 ul reaction volume; most often we use so-called "half reactions" of 3 ul DTCS Master Mix in a final volume of 15 ul. See our Sample Sequencing Form for a full explanation of the half reactions, and our Sequencing Flow Charts for quick one-page displays of all 6 parts of a typcial sequencing project (see side-bar); each part has its own Flow Chart with links to more detailed procedures and helpful information.
You need not follow our protocols for the half-reacion cycle sequencing reactions. Other sources for setting up your cycle-sequencing reactions include:
If you wish to follow our protocols, then start with the Sample Sequencing Form and read it carefully because we use half-reactons. Next, consult the Sequencing Flow Charts for both a general overview of the whole process (as displayed in the side-bar) and also for links to more detailed procedures and information, which can be found on each one-page flow chart.
We strongly recommend that you use the provided Sequencing Data Sheet (also available in the Olin Sequencing Lab) to set up your cycle-sequencing reactions. The sheet helps you to organize your reactions, to keep track of which ones go into which microtiter plate wells, and to trouble-shoot any potential problems before you start your reactions in the thermal cylcer.
|The protocols presented here are mainly for students in lab courses with sequencing exercises, and for research students and faculty members working on projects during the academic year and in the summer research program. Since several courses incorporate both the theory and practice of DNA sequencing and sequence-data analysis into their lectures and labs, we designed these methods to support such pedagogies. We therefore present the methods with details and explanations appropriate for student laboratory sessions. See also the actual lab session protocols from my lab manual (unpublished so far, but working on it): BIO 315 Molecular Biology Labs 9 & 10|
For an overview of the entire sequencing process (from template preparation to bioinformatic analysis) and with clickable links to the more detailed protocols and analysis programs, see our:
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