Sanger sequencing is provided on three ABI 3730xl DNA Analyzers. Clients may choose different levels of service depending on their needs.
Information on submitting samples can be found here: Submission Information. Data is recovered over the internet as described here: SangerComputerInformation.
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.
User provides quantified template (spec and gel recommended) and primer, we set up, cycle, clean, and analyze the reaction. Users are encouraged to provide enough material for at least 2 reactions in case a re-run is necessary. A reasonable attempt will be made to get good sequence in the presence of repeat regions and secondary structure, but additional reactions requiring additives will be charged extra. Good templates should yield 700-800 bp of sequence with a Q value >20 (standard of good data), with another 150-200 bp of OK sequence (technical jargon).
User provides 24 ul of template, primer, and water in individual 0.2 ml PCR tubes, corresponding to a double sequencing reaction (e.g., 400-500 ng plasmid template or 40-50 ng cleaned PCR fragment, 10-20 pmol primer). We will transfer 12 ul, add enzyme, buffer, DMSO, then cycle, clean, and run. Quantification of template by spec and gel is highly recommended. The additional 12 ul insures if there is a problem, we can re-run the samples without requiring more material. Tubes should be numbered, but sample names can be entered as anything, provided they correspond to file name syntax rules. A single sample qualifies for discount sequencing-striptube (below), but multiple samples must be in attached striptubes or they will be charged at the $10/sample rate.
Same as above, only reactions are provided in PCR strip tubes. Striptubes should be numbered, but sample names can be entered as anything, provided they correspond to file name syntax rules. Multiple single tubes are not allowed (they're charged at discount sequencing rate, above), nor are plates allowed (see full plate sequencing, below). Many companies sell striptubes, ours are from Axygen cat# PCR-0208.
Full Plate Sequencing
User provides a 96 well plate containing any primer and template in the appropriate concentration such that we have enough to do two reactions, total volume 24ul. If there is a problem with the first run, this insures that the user doesn't need to set up another plate. This is operationally similar to discount sequencing striptube, only in a 96 well format.
Cycled and cleaned up samples are provided to us in at least 20 ul of liquid in PCR striptubes (10 ul is withdrawn, we request 20 in case a re-run is necessary). 8 tube strips are preferred, but 12 tube strips are OK. Plates are not acceptable (see below for 96 well plate options), even if they are cut up, and single tubes are not acceptable (unless you have one sample). If you are doing magnetic bead cleanups, the beads can remain in the liquid. We strongly recommend bringing over at least 20 ul of sample. You can call the samples whatever you wish, following the punctuation restrictions, but identify the strips themselves with some numbering on the sides of the tubes; we also recommend putting your PI's initials (and yours if you wish) on them somewhere. Bring the strips over covered, and in a bag or wrapped up somehow to distinguish them as yours. While a little tape or parafilm to wrap strips is OK, please avoid massive wrapping. Also, most markers lift off with tape, so don't put tape over the critical info about your samples. The striptube option is the default ordering service. Samples in excess of 192 need to be entered on a separate order, and pricing starts over at 1-16 rate.
Single Lane Big Dye
Cycled, cleaned up, and dried samples are provided in 1.5, 0.5, or other tube formats. These samples will be resuspended and transferred to strip tubes by us eventually, so you may as well do it yourself and save yourself some money.
Big Dye Plate
User provides 96 well plate ready for running, operationally similar to having 96 striptubes. If sample names other than well positions are desired, user can upload an Excel spreadsheet containing whatever file names are desired using this Excel spreadsheet template. Note that plates are run a certain way so be sure your names follow the appropriate format by talking to us first.
Striptube plus Cleanup
Cycle sequenced samples are brought over in attached 0.2 ml PCR striptubes, no oil. Sample running charges go along with the Striptube rate schedule.
Single Lane BD plus Cleanup
Cycle sequenced samples are brought in loose tubes, either PCR or 1.5 ml microfuge tubes. Cleanup is done by magnetic beads, no clean up discount for multiple samples. Running charges go along with the Single Lane BigDye rate schedule. To get a better rate, consider transferring to striptubes.
Big Dye Plate plus Cleanup
User provides 96 well plate with cycle sequenced samples, no oil, that needs the unincorporated dye removed.If sample names other than well positions are desired, user can upload an Excel spreadsheet containing whatever file names are desired using this Excel spreadsheet template. Note that plates are run a certain way so be sure your names follow the appropriate format by talking to us first.
RTG BigDye Plate
User provides half skirted, foil sealed plate (we provide plate and seal) containing appropriately aliquoted amounts of cleaned up sequencing reaction (10 ul sample plus 10 ul water, or 20 ul straight sample if signal strength is a concern). No magnetic beads allowed, please. If sample names other than well positions are desired, user can upload an Excel spreadsheet containing whatever file names are desired using this Excel spreadsheet template. Note that plates are run a certain way so be sure your names follow the appropriate format by talking to us first.
User provides at least 10-15ul of each cleaned up 96 samples in either 0.2ml arrayed striptubes or plate. Molecular weight ladder (please inquire which ladders are available) will be added to 2ul sample. If sample names other than well positions are desired, an Excel spreadsheet can be uploaded containing the custom file names using this Excel spreadsheet template. Note that plates are run a certain way so be sure your names follow the appropriate format by talking to us first. For more information please view this site, Fragment Analysis.
RTG Genescan Plate
User provides half skirted, foil sealed plate (we provide plate and seal) containing appropriately aliquoted amounts of molecular weight ladder and sample in high quality deionized formamide to a minimum volume of 12ul. Please let us know the dye set to be used. If sample names other than well positions are desired, an Excel spreadsheet can be uploaded containing the custom file names using this Excel spreadsheet template. Note that plates are run a certain way so be sure your names follow the appropriate format by talking to us first. For more information please view this site, Fragment Analysis.
Full Plate Run
User provides a 96 well plate containing PCR amplifiable material. This could be genomic DNA, or a plasmid library in a standard vector (i.e., primers on either side of the insertion site are available) with inserts less than 3-4 kb in length. We return 96 sequence sample files representing sequence from one side only.
Base cost, one direction only: $650
One 96 well plate, sequenced both ways (192 files): $1000
Some of the above options are billed specially, not entered from the computer (come talk to one of us).
If you do not have a full plate the cost is as follows:
For sequencing in one direction only, the cost is $12.00 each for the first 16 samples, then $9.50 each for the next 48. Anything above that runs at $650.00 for the full plate. As an example, for 36 samples that would amount to ($12 x 16) + ($9.50 x 20) = $382.00.
For sequencing in both directions, the cost is $18.00 each for the first 16, then $14.50 each for the next 48. Anything above that runs at $1,000.00 for the full plate. For 36 samples that would amount to ($18 x 16) + ($14.50 x 20) = $578.00.
We can also set custom prices for projects not included above. Our charges will be as close to cost as calculable. Just ask us about services you don't see covered in this document.
A number of service options for DNA sequence determination are available through our facility. The most economical service involves the user setting up their own reactions, then bringing the cycle sequence products to our facility. We can provide the sequencing reagent at a very low rate thanks to a favorable University quote, and go over the protocols with you necessary to carry out the reactions in your laboratory (a PCR machine is the only required equipment). Various sequencing protocols that have worked for different groups can be found in this document: "Cycle Sequencing Protocols."There is a another document describing the particular protocols we use in the facility, called "Facility Procedures." We also can provide positive controls to help you get started (as described in the document "Available Controls" at the DNA files page, or troubleshoot when results are not satisfactory.
We also can provide expert service in sequencing if the user wishes simply to provide the template DNA. We can sequence double strand plasmid, single strand phage, PCR products, BACs, cosmids, and related vectors. Common vector primers are provided (e.g., SP6, T7, M13F and M13R, etc.-check availability). Every effort is made to provide at least 600 bp of excellent sequence (Q value >20), though template composition and purity are crucial determinants of quality. Strategies are available to sequence difficult GC rich templates or homopolymer regions, user should indicate if such areas are present.
The DNA sequencing facility can also carry out larger sequencing projects involving editing and assembly. The maximum size of the sequence we can generate is approximately 20 kb. Price is based on the degree of confidence required (i.e., single strand vs. double strand, one vs. multiple passes); check the price list or call for more information.
Finally, a number of different options are available for the processing of 96 well plates. Please consult the price list to see what we can do for you if samples are in, or could be generated, using this format.
The UWBC DNA Sequence facility can also work with researchers to carry out services that may not be listed in our current rate schedule. We can generate custom charges based on our estimates of what the work will cost. If you don't see something you want us to do, please inquire whether we can carry out this service.
Note: non-UW users are charged 55% above the rates listed below