ORDER FORM | RETRIEVE YOUR DATA | Description | Pricing | Oligo Calculator | FAQ
FREQUENTLY ASKED QUESTIONS:
Q. When I open up my electopherograms, all I see is a string of Ns. What's wrong?
The symbol N means that the computer analyzing the data could not call the base (or bases).
A string of Ns and no other data means that the sequencing reaction produced no data. See
the following information about possible reasons for sequencing failures.
Q. Why did my sequence fail?
Here are some common reasons:
Poor quality template DNA. The quality of the DNA template is critical to getting good data. Common contaminants include:
- Residual salts and organic chemicals (e.g., phenol, ethanol)
- RNA
- Proteins
- Excess PCR primers and reagents from prior PCR reactions
Not enough DNA. For double-stranded plasmids, a DNA concentration of 80-120 ng/ul is required. We rely on your estimate of template DNA concentration. In general, the more accurate the estimate, the better the quality of data that will be obtained. Too much DNA can be almost as bad as too little. We suggest that you measure the DNA concentration by comparison with a known amount of DNA on an agarose gel or on ethidium bromide/agarose plates.
Not enough primer. We require a primer concentration of about 2 µM, which for a 17mer is equivalent to a about 20 ng/µl.
Primer or template was not added to the reaction. Occasionally, either the primer or template is accidentally left out of a reaction.
Degraded Primers Although primers are generally stable, repeated freezing and thawing can lead to degradation of the primer. We recommend freezing a stock solution of primer, and using smaller aliquots for a working solution.
The Tm of the primer is < 50 °C. The annealing temperature in our cycle sequencing reactions is 50°C, Therefore, all primers should have a melting temperature of 50-55°C. The melting temperature of a primer should be determined by a program (Oligo® or Primerselect from Lasergene) that uses the nearest neighbor summation of thermodynamic parameters to calculate an accurate Tm. We recommend that sequencing primers be between 18 and 30 nucleotides in length.
The template does not contain a sequence complementary to the primer. For example, the pGem plasmid vectors do not contain a T3 primer site, but instead contain an sp6 site. Occasionally, annealing sites are altered in the cloning process; as a result, the original primer may not anneal.
Occasionally one of the primers used to generate the PCR product will not work in fluorescent cycle sequencing. It is likely that such a primer, although sufficiently competent in the exponential PCR process, is very inefficient in the linear amplification of cycle sequencing.
For more troubleshooting information, see the Automated DNA Sequencing Guide, Section 7-39.
Q. How much plasmid DNA do you need for a standard premixed tube reaction?
We require 600 ng - 840 of plasmid template for each sequencing reaction you submit. If you have a large plasmid, i.e. >8kb, use the higher amount of DNA. Add 2 pmoles of primer to that and submit it for sequencing. Each tube you submit should contain a total of 8 ul: 6 ul of DNA and 2 ul of primer. Use only ONE primer for each sequencing reaction.
Q. How much PCR product do you need for a standard sequencing reaction?
The amount of DNA required depends upon the size of the product. The smaller the PCR product, the less DNA we need to obtain a strong sequencing signal. Conversely, sequencing large products requires more DNA. See Guide to Sequencing PCR Products. for detailed information.
Q. I forgot the password to my web site data folder. What should I do?
Send an e-mail request to have your password re-set to ejung@wfumb.edu or kkourman@wfubmc.edu. Once the password has been reset, you'll receive an e-mail message that notifies you of the change, and instructs you to change your password.