What if my DNA is longer than your machines can read?
You say your machines can read sequences up to 800 or 1000 nt long. What if my DNA is longer? How can I determine the sequence for the whole thing?
The U of M DNA Sequencing Core provides only raw sequencing. If you provide a template and a primer, we will return the results of *one* sequencing reaction (typically 800+nt for good templates).
**** We do not perform primer-walking or shotgun sequencing. ****
If you need the sequence of a larger fragment, please see the following flowchart to determine your options.
If you need a complete sequence from a larger fragment, here’s how to proceed:
- Are you looking for mutations in a known sequence?We recommend that you design primers all at once that will cover the entire length of your insert. See our web page on designing sequencing primers. Have all your primers synthesized at once and submit them to the Core for simultaneous sequencing.
For assembly of the final sequence from the individual chromatograms (note: NOT the text sequence, but the chromatograms!) we recommend the program ‘Sequencher’ from Gene Codes, Inc. We provide copies of this software for checkout at our MSRB II and NCRC locations.
- Is your insert completely UNKNOWN, and just a few kb in length?We recommend Primer Walking to get your complete sequence. The Core can perform only some of the steps necessary in this procedure (click the link Primer Walking to learn more). You may also want to see our discussion on designing sequencing primers.
- Is your insert completely UNKNOWN, and GREATER than 10 kb in length?We recommend shotgun sequencing to obtain your complete sequence. The Core can perform only some of the steps of this procedure (click the link shotgun sequencing to learn more).
- If your UNKNOWN sequence is greater than a few kb, but less than 10 kb, you fall into an intermediate range. Primer walking will take a long time, but shotgun sequencing is a bit of overkill. Both will work, but you’ll need to choose between those two options, based on what you are comfortable with.