Sanger Sequencing: Sample submission information
Numerous formats are available for sample drop off, depending on what service is requested. For samples that have already been cycle sequenced and need to be analyzed by gel or cap electorphoresis, different formats and numbers of samples result in different charges. Please consult our Sanger Prices for details.
For samples to be dropped off for sequencing, the following guidelines are recommended for best results and shortest turnaround time. If the template DNA is a plasmid <20 kb in size, it should be at a concentration between 100-200 ng/ul, with enough sample dropped off to repeat the sequencing if necessary. Very dilute samples (10 ng/ul minimum concentration for plasmids) and very concentrated samples can also be dropped off but may affect results and turnaround time. Ideally the primer should be dropped off at 5-10 pmol/ul concentration (5-10 uM, or 30-60 ng/ul for an 18mer) with enough to do several reactions in case we need to repeat the sequencing. Most plasmid template prepared by columns (from a number of manufacturers) can be expected to give 600 bp of very good data and an additional 200 bp or so of decent data. We recommend quantifying DNA by spec AND agarose gel. Larger templates such as cosmids and BACs should be at an approximately 5 fold higher concentration for submission. If the template is a PCR fragment, additional cleanup by dilution (~20 fold) is the only thing that needs to be done following the PCR reaction and before cycle sequencing. This cleanup method is limited to PCR reactions that give a single major band. Gel purified fragments can be used as well. The dogma on amounts to use in sequencing for PCR fragments is 10ng/100 bp, but it is difficult to quantify PCR fragments so sometimes a gel picture is sufficient for us to decide how much to use. Amplifying primers usually work well for sequencing.
Every effort is made to generate good sequence, including free re-dos often. Heroic measures for difficult templates (for example, extreme GC rich or homopolymeric regions) might cost extra and no guarantee is given. Similarly, BACs and cosmid sequencing is highly dependent on template purity so good data is sometimes problematic. We are happy to discuss further these issues with you. The more information we have about your template the greater chance to get good data in the shortest time. You must indicate the template and primer concentrations, whether it is a plasmid, BAC or PCR fragment, and whatever other information might be helpful to us prior to sequencing (for example, plasmid size or GC content, if these are relevant issues).
If you are shipping samples from off campus, the address is:
UWBC DNA Sequencing Facility
Rm 2360 Genetics/Biotech Bldg.
425 Henry Mall
Madison, WI 53706
Pad those samples! We have occasionally received crushed 1.5 ml tubes. Samples can be shipped wet or dry.
Every effort is made to provide
our users with the best data in a timely fashion for the advancement of
their research. Turnaround time is typically one working day. If you haven't
seen your data on the server by three working days, contact us. Samples
that have failed due to gel or capillary problems will of course be rerun
free of charge. Usually such problems have a characteristic appearance,
so contact us to see if this is where your difficulty might be. Samples
are kept seven days, so you must let us know within that time frame if
your samples need to be rerun. Our facility handles 10-15,000 samples/week,
and despite our best efforts we occasionally make mistakes. If you get
data that is completely puzzling (e.g., sequence from an organism that's
not even in the same phylum as your model system) please contact us as
soon as possible and we can re-run the samples. As above, we need to know
within seven days so we can rerun the same samples you brought over. Sometimes
we can even trace the problem and give you the correct sequences right
away. If the questionable samples are gone, the initial analysis and reanalysis
will be done free of charge, and a reasonable attempt made to compensate
for reagents wasted.