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Frequently Asked QuestionsBasic Information
How to prepare samples for sequencing Submitting your samples After you've submitted samples About your results Regarding the billing of our services If you have further questions contact Michelle Zanche 276-9997 What is DNA Sequencing?
Who can submit samples to this Core? Why use automated sequencing? How do I sign up? Does the Core do Genotyping as well? What types of templates can the Sequencing Core process? What will it cost? Can I get bulk discounts for large-scale projects? What will I get for my money? We haven't used the Sequencing Core in a long time. What do we do? How to prepare DNA
What primers does the Core provide? How do I design my own primers? The correct concentration and volume for your template(s) and primer(s) What are my choices do I have for submitting my samples?
How do I log my samples in? (How do I get a sample number?) How do I label my samples? Where do I drop off my samples for the Core? How do I check the status of my samples? I made a mistake in entering my samples. What do I do? How long will it take?
How do I check the status of my submitted samples? How will I get my results back? How do I contact the Core personnel? How do I download my chromatogram file?
How will these services be billed?
How do I open my chromatogram file? What do I do in case of problems? What are the most common reasons for a sequencing reaction to fail? How can the Departmental Financial Contacts resolve accounts? Basic InformationWhat is DNA Sequencing?DNA Sequencing is a process, usually involving electrophoresis, that reveals the order of the four nucleotides in a fragment of DNA (deoxyribonucleic acid). This is done by labelling each nucleotide (A, C, G or T) with either a radioactive or fluorescent marker which identifies it.How is DNA sequencing done?Chromosomes, which range in size from 50 million to 250 million bases, must first be broken into much shorter pieces (subcloning step) Each short piece is used as a template to generate a set of fragments that differ in length from each other by a single base that will be identified in a later step (template preparation and sequencing reaction steps) The fragments in a set are separated by gel electrophoresis (separation step) New fluorescent dyes allow separation of all four fragments in a single lane on the gel. The final base at the end of each fragment is identified (base-calling step). This process recreates the original sequence of As, Ts, Cs, and Gs for each short piece generated in the first step. Automated sequencers analyze the resulting electropherograms, and the output is a four-color chromatogram showing peaks that represent each of the four DNA bases. After the bases are "read," computers are used to assemble the short sequences (in blocks of about 500 bases each, called the read length) into long continuous stretches that are analyzed for errors, gene-coding regions, and other characteristics.Who can submit samples to this Core?The Nucleic Acid Core handles clinical and research samples, from the university community, other educational, as well as outside industry.Why use automated Sequencing?
How do I sign up?In order to sequence your samples we require that you first set up an account on our sever. You will need some basic information on you lab in order set up the account. Such items would be: Who is your PI (department, phone, email and address): who are you (name, address, email): what account(s) will sequencing be billed to.Does the Core do Genotyping as well?Please see our information on our Genetic Analysis pages.What types of templates can the Sequencing Core process?Plasmid and PCR Products.What will it cost? Can I get bulk discounts for large-scale projects?Please see our information on our service pricing page.What will I get for my money?Our sequencers typically give 500 BP read lengths for good DNA samples. We will repeat any samples that fail due to problems in our own procedures or instruments, but we do not accept responsibility for poor results arising from improperly prepared templates or primers,or from improperly designed experiments.We haven't used the Sequencing Core in a long time. What do we do?What you need to do is bring our records up to date to start sequencing again. Undoubtedly some of your personnel and account records are out of date, plus there have been some changes in the Sequencing Core's operations that require you to do some minor upkeep. If you don't already have an account on our LIMS system please log on and create a new account. If you currently have an account please make sure the information is all current.How do I sign up?First you need to create an account on the server. (http://fgclims.urmc.rochester.edu/) The first time you connect to our server, you will need to enter some basic information. For example: who is your PI (department, phone, email and address); who are you (name, address, email); what account(s) will sequencing be billed to. Once your account is created you are ready to submit your samples.How to prepare samples for sequencingHow to prepare DNAPlease see our recommendations for how to prepare your samples for processing in our facility.What primers does the Core provide?The Core facility provides the following primers:
How do I design my own primers?Our facility offers software to help you design your own primers. Located in our lab are 2 public data analysis workstations that contain this software. To sign up for time on our public workstations please see our calendar.The correct concentration and volume for your template(s) and primer(s)Using the Premixed Sequencing Form on DnaLims to register your samples, bring us your purified templates and primer in 1.5 ml eppendorf tubes as below:
Submitting your samplesWhat are my choices do I have for submitting my samples?We offer several options for submitting your samples. Please view our page on submitting your samples.How do I log my samples in?The DNA Sequencing Core uses its computer systems to track all samples, prioritize their processing and return the results to the investigators by a website. In order to process your samples they first must be registered in our system. You can do this by logging into the system and entering your samples names. Please keep the naming of your samples simple and be sure that your tubes are labeled appropriately.How do I label my samples?Our staff handles hundreds of samples a day. To ensure accuracy we ask that you please be sure that all of your samples are clearly organized and labeled.Where do I drop off my samples for the Core?Please drop off your samples in the appropriate refrigerator that is located in the Medical Center ground floor room G-7814 (Near Photography ). Make sure all your samples are labeled clearly and have a copy of your sample submission sheet from our system.How do I check the status of my samples?When your samples have been processed you will receive an e-mail confirmation asking you to login to dnaLIMS.I made a mistake in entering my samples. What do I do?Login to dnaLIMS. Click on View or Delete a Sequencing Request. Click on your order number and delete the request or sample number you wish to remove.After you've submitted samplesHow long will it take?Dried down samples submitted before noon will take 24-48 hours. All other samples submitted before noon will take 48-72 hours.How do I check the status of my submitted samples?You can Log on to dnaLIMS at any time to view the status. If your results have not been posted after 72 hours, please contact the core facility at 276-9997.How will I get my results back?Your results will be posted to our site when they are complete. You will be notified via email when they are ready.How do I contact the Core personnel?Please contact the core staff either by email at Michelle_Zanche@urmc.rochester.edu or phone 276-9997.About your resultsHow do I download my chromatogram file?Once your sequencing is complete and you have received an e-mail notification from the Core Facility, please go to the following site and log in:http://fgclims.urmc.rochester.edu Once you have logged in click on the Retrieve Sequencing Result button, select the desired order number, and click on the Submit button. If you only care to view the text file it will open up in Notepad from this site. If you would like to view the chromatogram version of your sequencing files you must first download them using the Chromate button at the top of the page. How do I open my chromatogram file?Once your chromatogram file is downloaded, go back to the menu page, and click on the Support Tools button. This will link you to a page where you can download the appropriate programs for your computer:
If you are using a PC: Open the Chromas application first, then open your file through Chromas. (You will typically place the downloaded file in the same folder as your Chromas application.) If you are using a MAC: Drag your chromatogram files on top of the repair tool icon. This will convert the files so that the MAC recognizes them. Now open Editview first, and view your files through Editview. What do I do in case of problems?If you have questions or concerns about your sequencing please contact the core lab staff.What are the most common reasons for a sequencing reaction to fail?In short, poor template and/or poor primer design. Please see our troubleshooting page.Regarding the billing of our servicesHow will these services be billed?The services or products are billed through our point of sale computer by using your 10 digit university account number. An invoice is mailed to you via intramural mail. Please be sure that the person in your area doing your bookkeeping gets the invoice.How can the departmental financial contacts resolve accounts?Any questions related to the purchase of products or services with our lab should be directed to our Staff Accountant Dave Terry via email dave_terry@urmc.rochester.edu or by phone at 276-9998 |
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