DNA SAMPLE PREPARATION
The length of read is proportional to the quality of the DNA template and the efficiency of a particular primer. It is essential to provide templates of a very high standard, free from contaminants such as salt or ethanol. Read lengths of 1kb and above with excellent base calling are achievable with high quality templates.
DNA Sample Quality
We recommend using a good quality cleanup kit. Qiagen and Promega kits have proved reliable, but there are many alternatives currently available. Care should be taken to avoid overloading columns with very highly concentrated samples, as this can compromise the efficiency of the cleaning process. Particular care should be taken to ensure that no traces of ethanol are carried over into the DNA sample as this causes inhibition of the sequencing reaction. We recommend eluting in water rather than the provided buffers, as some buffers also inhibit the sequencing reaction.
If providing PCR products for sequencing these should be cleaned using an appropriate kit to ensure complete removal of residual dNTPs and primers, and should be eluted in water rather than the supplied buffers.
DNA Sample Concentration
Initial estimation of DNA concentration is vital to ensure successful sequencing. Although all DNA samples are anaysed on agarose gels upon arrival, we strongly recommend customers check the concentration of their samples before dispatch to ensure they fall within the following recommended guidelines. We have found agarose gel estimation using concentration standards to be much more accurate than nanodrop or spectrophotometry as contaminants in the DNA solution can affect the OD reading, leading to a false high concentration. As a general guideline for plasmid preparations, run 1μl on an agarose gel and if you can't see it the concentration is probably too low for sequencing. It is possible to obtain sequence data from templates with low concentrations, but the quality of the data depends on the quality of the template DNA. Remember to send a little more DNA than is required, so that we can confirm the concentration before we prepare your reactions.
TOTAL DNA REQUIRED PER REACTION
|Plasmid DNA||10-250 ng/μl||125 ng|
|Large templates||100-500 ng/μl||500 - 100 ng|
|PCR fragments||10-250 ng/μl||5 ng per 100bp of product|