My reaction didn’t work! What could have happened?
It is always disappointing when one’s reaction does not go as planned. Poor sequencing results or a ‘flat-line’ non-existent reaction can be due to several factors. As a first step, please review the information below and in our FAQs. Common reasons for reaction failures are detailed in these sections of our website and will probably allow you to resolve your issue. However, if you are still experiencing issues or need more advice, please do contact us so that we can help you further.
Additional details regarding interpretation your results can be found on the Retrieving/Understanding your Sequencing Results page
Template Quality and Quantity
In order for a sequencing reaction to yield robust results, it is critical that the template used for these purposes is of high quality. Commerical kits routinely yield such high quality DNA.
If you are not using a kit, DNA template derived from such methods as ‘boiling’ will not yield template of sufficient quality and we discourage users to submit samples purified in this manner. However use of a standard alkaline lysis method followed by clean-up of the resulting DNA using a deproteinating step (e.g. phenol/chlorofom or equivalent) and ethanol precipitation would yield template of the appropriate quality.
As always, we are happy to save you a step by purifying your DNA from your miniprep or maxiprep.
Template Makeup
We can sequence most common types of DNA, including plasmids, single-stranded phage, PCR products, Lambda, BAC, PAC, and YAC DNA. For a list of the types of DNA we can sequence and amounts required, please follow the services link at the top of this page to learn more.
Other considerations include the extent of GC content and template size. Please contact us to discuss any special considerations regarding your sequencing reaction which you are concerned about.
All types of DNA to be sequenced must be supplied in Milli-Q water, since EDTA, a component of Tris-EDTA (TE) buffer, can interfere with the sequencing reaction.
Primer Quality and Quantity
We encourage customers to review our Suggestions for Optimal Sequencing Primer Design