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:

http://seqcore.brcf.med.umich.edu/doc/dnaseq/primers.html

Preparing Templates:

http://seqcore.brcf.med.umich.edu/doc/dnaseq/dnaprep.html

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.

For a more detailed list of templates see the website below:

http://seqcore.brcf.med.umich.edu/cgi-bin/concentration.pl?templatetype=all

Direct Sequencing of PCR Products:

http://seqcore.brcf.med.umich.edu/doc/dnaseq/pcr.html

Submitting your Samples:

Go to: http://seqcore.brcf.med.umich.edu/AllSamples.html

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:

Go to: http://seqcore.brcf.med.umich.edu/ftpdata.html

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