Proper DNA quantification is an often overlooked, but important issue to consider before submitting samples. The minumum and sugested volumes to be used in the reaction are displayed in the right hand column of the review page when ordering. Always send double the amount of template to allow for evaporation, or three time the amount of template so that reactions that do not work initially may be reprocessed.Please see our Sanger Sequencing Troubleshooting Guide - What you need to consider when preparing your template section for additional information. Make sure that the templates you are submitting are accurately quantified, salt/contaminant free, and clean.TE interferes with the sequencing reaction and will produce poor results. Re-suspend templates and primers in sterile dH 2O ONLY - DO NOT dilute samples in TE.Send all templates within the same 96-well plate at a similar concentration.The presence of excessive amounts of template, protein contaminants or carbohydrates can coat the capillary walls resulting in poor data resolution. salts, proteins, detergents like SDS or Triton X-100 and degraded RNA fragments) contaminating your samples will compete with the DNA during injection causing a reduction in signal strength. When the samples are being transferred into the capillaries, any ions (e.g. The electrokinetic injection system of the sequencer is highly sensitive to salt and other ions that may be in your samples. The quality of DNA templates is key to good sequencing results. Once your samples on placed on a run, your order can not be canceled. Clients needing to cancel an order must contact our facility at kevcav (at) iastate (dot) edu before we place your samples on a run.We recommend that you retrieve your results as soon as it's available. Once your order is complete, you will be notified via e-mail that your data is ready for download.Orders pending the receipt of samples are kept in the OnCore receive queue for approximately 3 weeks.Additionally, clients should contact us via e-mail at kevcav (at) iastate (dot) edu to let us know that you have entered a new order using samples currently at the facility and you will not be sending additional samples. Clients using samples from a previous order should include the corresponding tracking number in the comments section of their new order (e.g.Templates and primers are stored at the facility for approximately 3 weeks.Initial Sanger sequencing results are typically posted within 24 hours after receipt (not counting weekends, university holidays, or orders requiring primer synthesis).Please remember to include your OnCore tracking number with all correspondence and materials shipped to the facility. Submitting Samples for Standard (Single Tube) Sequencing Optimization methods can involve altering BigDye chemistry, changing thermocycling conditions, changing primers, or combining techniques. Our Full-Service option includes reprocessing if we feel that the initial sequencing yielded poor results or optimization of reaction conditions could potentially improve sequence length or quality. Pricing for this service includes either a Full Service or a No Redo option. Our standard Sanger sequencing allows for the highest flexibility in primer-template pairings and can better handle difficult templates or variations in DNA concentration. Please review our " fees for service" and " terms of service" pages before placing your order. Your samples can be sequenced using either facility stock primers, custom primers sent with your order, or custom primers synthesized at the facility. Our Sanger DNA sequencing services can process plasmid, PCR products, cosmids, phage, BAC, and bacterial genomic DNA samples in either a single tube or 96-well format. Sequencing on the AB 3730xl allows clients maximum flexibility in read length and number of samples. The AB 3730xl is a fully automated system that detects fluorescently labeled DNA fragments and can provide up to 800-1000 bases of high-quality DNA sequencing data per reaction. Both standard and high throughput Sanger DNA sequencing samples are run on the facility's two Applied Biosystems 3730xl DNA Analyzer. The DNA Facility's Sanger DNA sequencing services are optimized for the rapid turnaround of high-quality sequence data for individual samples or high-throughput plate sequencing.
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