Does the Core perform Shotgun sequencing?
Can the UM DNA Sequencing Core perform shotgun sequencing?
The University of Michigan does not at this time offer a shotgun sequencing service. We can, however, help you in certain aspects of the procedure, if you want.
Here are your options:
- Send the sequencing job to a company that can do complete shotgun projects.
Talk to the Core Director (Dr. Robert Lyons) regarding suggested companies that may be able to help you.
- Get the shotgun library and DNA preps done elsewhere, then have the Core do the actual sequencing.
Several groups have done this, and it works well. Again, talk to the Core Director (Dr. Robert Lyons) for suggestions.
- Make shotgun libraries yourself, purify the plasmids, and submit them for sequencing.
Here’s a brief description of the steps in making a shotgun library for your unknown DNA:
- Isolate your unknown DNA in relatively pure form.
- Fragment it RANDOMLY into pieces of 500-1000 nt. Consider sonication or similar.
- Form blunt ends (e.g. Klenow treatment).
- Add a single 3′ A (e.g. with Taq polymerase).
- Clone the fragments into a typical general-purpose T/A cloning vector.
- Pick and grow perhaps 2-3 clones for every kilobase of unknown sequence you need.
- Isolate plasmid, submit to for sequencing with vector primers from both ends.
- Assemble the sequence. We recommend using the ‘Sequencher’ software from GeneCodes, Inc. for projects of up to several tens of kb.