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DNA Sequencing Protocol

Christa Van Dort (Updated April 15, 2005)

The University of Michigan's DNA Sequencing Core has an excellent website that is very extensive and can be found at: http://seqcore.brcf.med.umich.edu/
Go to 'About the Core' for most questions.
 
Designing Primers:
 
Preparing Templates:
 
Template and Primer Concentrations:
Each Sequencing Reaction needs 10 µL of template and 10 µL of primer in Separate Tubes. If one sample (template or primer) is to be used in multiple reactions, multiply the volume accordingly.
Template Type Template Conc. Primer Conc.Notes 
Plasmids (mini-preps, maxi-preps, 4-15kb200 ng/µL3.2 pmol/µL  
PCR Products (rule of thumb)For each 100 bp of fragment size, 1 ng/ul
For Example:
500 nt 5 ng/µL
3.2 pmol/µL Remove old PCR Primers! 

For a more detailed list of templates see the website below:
 
Direct Sequencing of PCR Products:
 
Submitting your Samples:
Or click on Submit Samples from the Core homepage
The PI login is: macdougald
The user login is: usually your last name
Once you have entered your samples online, label the top of 1.5 ml eppendorf tubes with the corresponding numbers they give you.
Take your samples to the DNA Sequencing drop off desk located at 2560 MSRB II
Their hours of operation are 8:30am – 12:15pm and 1:15pm – 4:45pm
 
To get your chromatograms:
Or click on Download Results from the Core homepage
Enter the PI login: macdougald
In the box that comes up enter the lab password: 76206
Note: You must have a special program to view chromatograms, which can be downloaded from http://seqcore.brcf.med.umich.edu/usechrom.html#program