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DNA Sequencing FAQ

Services

General Information

Sample Preparation

Sample Submission

Receiving Results

Still have questions?

Contact:
Lisa Bukovnik
lisa.bukovnik@duke.edu
919-660-7436
119 Biological Sciences Bldg. Science Drive
Durham, NC 27708

Services

What services are available at the IGSP Sequencing and Genetic Analysis Facility?

The DNA Sequencing Facility Offers the Following Services and Prices

SERVICES

  • Dye-Terminator Removal (DTR) and Electrophoresis of Client-Prepared BigDye-Terminator (v.3.1) Reactions
  • Large-Scale Sequencing using Primers Chosen by the Client
  • PCR Cleanup in 96-well Format
  • Microsatellite (post-pcr) Dilution, Pooling,and Electrophoresis
  • AFLP (post-pcr) Dilution and Electrophoresis
  • Sale of BigDye Terminator v3.1 and its buffer at Cost
  • Sale of PCR Plates at Cost
  • Troubleshooting of Sequence Reactions
  • Methods Development on Biomek FX 96-Channel Liquid Handling Robot

PRICES
As of February, 2006, the following prices apply:

  • DTR and Electrophoresis: $1.50 per reaction.
  • Microsat. or AFLP (500 size standard): $98.00 per plate of 96.
  • Microsat. or AFLP (1000 size standard): $105.00 per plate of 96
  • PCR Cleanup: $20.00 per plate of 96.
  • BigDye v.3.1: $335.00 for 400μl. Buffer cost: $15.00 for 400μl
  • PCR Plates: $50.00 per box of 25 nonskirted, labeled plates.
  • Tips for special robotics projects: $6.00 per box.

General Information

Who can submit samples to the IGSP Sequencing Facility?

All Duke researchers with valid Duke email address and fund code may use the facility. Clients outside Duke are welcome on a limited basis due to accounting regulations, and they are charged $8 per data sheet and a $15 monthly maintenance fee in addition to the $1.50 per sample fee.

How do I register for a sequencing account?

Each LAB needs a partition on the sequencing server, bio-lemming, so its members can receive sequence data in a secure electronic form. At that point each LAB MEMBER will have access to the "DataSheetDropoffFolder." This folder is used by the individual researcher to leave an spreadsheet containing information for me about your samples. You will also be added to an email list used for announcements concerning the facility.

The first thing you need to do to get started is ask your fellow lab members if your lab has a partition on the bio-lemming server partition DNA_Seq for sequencing. If your lab does have an account, ask your lab manager for the access instructions. If your lab does not have an account, email me (lisa.bukovnik@duke.edu) and I will get you started.

How do I initiate a sequencing project?

Please download this zip file and follow the instructions inside. Put your data sheet in the DataSheetDropOff folder. This alerts me to expect samples from you. You are responsible for doing your own cycle sequencing: you quantify your template, choose your primer, add the right amount of BigDye v 3.1, and do the cycle sequencing.

Then put your data sheet, properly filled out, in the "DataSheetDropoff" folder on bio-lemming. This lets me know to expect samples from you. Bring your reactions to my lab and I will do the post-reaction DTR (dye-terminator removal) and the electrophoresis, then I will put your data in your lab's data folder. I do not screen your data, but I can help with trouble shooting.

What is the cost of sequencing a sample?

For Duke researchers, the cost for DTR and electrophoresis is $1.50 per reaction. Non-Duke researchers are accepted on a limited basis for accounting reasons, and are charged $8 per data sheet and a $15 monthly maintenance fee in addition to the $1.50 per sample fee.

Where is the IGSP DNA Sequencing Facility located?

The lab is located in the Biological Sciences Building on Science Drive, West Campus, Room 119. The phone number is 919-660-7436. Office hours are 9:00 am - noon. A 24hr drop off is located at BioSci 123.

Sample Preparation

How should I prepare my samples for sequencing or electrophoresis?

After you get access to the bio-lemming server, you can start sequencing. There are a few things to know about how your samples are handled here, which might be different from the way you've had them done in the past. First, YOU do your own cycle sequencing reaction (quantify your template, choose your primer, add the bigdye and run the rxn. in the thermal cycler).

After that, bring your reactions to me (BioSci 119) where I will do the post-reaction DTR (Dye-Terminator Removal) and electrophoresis on my ABI 3730 xl' s. For more detailed information about how to do the reactions themselves, consult the BigDye manual at the Applied Biosystems website.

Follow the protocols listed there, doing a one-eighth strength reaction.

Run a control. OR you can start with the reaction protocol listed here, and optimize for your situation. RUN A CONTROL. SQ PROTOCOL

How much DNA is required for sequencing?

The University of Michigan DNA Sequencing Core has done a great job summarizing template concentrations. Click here for more information.

Where can I get the BigDye and buffer?

BigDye ver. 3.1 and its 5X Dilution Buffer are available for sale at cost in my lab in aliquots of 400 UL.

Bring something to keep it chilled if you have a long walk back to your lab. Look in the small freezer in between the two capillary sequencers in BioSci 119, and take the NEXT CONSECUTIVELY NUMBERED TUBE. Look carefully to get the right number! PUT YOUR FUND CODE on the clipboard on top of the freezer. This is how we know where to send the bill.

The BigDye buffer is next to the big BigDye rack. Use this same signout procedure.

Sample Submission

How should my samples be labeled?

Please follow these instructions very carefully. I process sometimes thousands of samples per day, and I do not want to get yours mixed up with anyone else' s!

Labeling: For tubes, put your tubes in a microtube rack with your lab' s name written with Sharpie pen on all four sides of the rack. Stick a piece of tape on the rack, or on top of the caps. On the tape, write your name, email, lab, date, and total number of samples in the rack. The name on the tape needs to match the name on your data sheet.

Samples are processed and data are distributed according to the individual, not the lab. That's why I need YOUR name on the rack as well as the lab name.

For plates, put the tape on top of your seal. Indicate the location of sample A1 with an asterisk in Sharpie. Sample 8 should be at position H1, sample 9 at position A2, sample 16 at position H2, etc. DO NOT SUBMIT PARTIAL PLATES FOR FEWER THAN 24 SAMPLES.

Pick up your lab's empty racks in Room 123.

What tubes or plates may I use?

Make your samples robot-ready.

The Seq. and Genetic Analysis Lab processes thousands of samples daily with the aid of a 96-channel liquid handling robot. Samples must be in uniform tubes or plates. Do not submit any low-profile materials. Do not submit strips of tubes with tabs on the ends. USE NO HINGED CAPS!! Use removable strip caps on your strip tubes.

1. Use tubes that come in connected strips of 8 with matching connected strips of completely removable caps. The caps need to be COMPLETELY REMOVABLE (not possible with hinged caps) for the robotic handling your samples receive. DO NOT put strip caps on hinged tubes with the caps cut off.

2. If you have fewer than a multiple of eight reactions, with scissors or a razor cut the strips of both the tubes and caps. NUMBER YOUR TUBES ON THE TUBES, NOT THE CAPS. The caps are THROWN AWAY; and I would like to keep your tubes the way you have them arranged. I will charge for empty tubes in a strip. Please trim them off!

3. Plates of 96 must be non-skirted, normal-profile plates. (You can buy them in the lab. See below.) Arrange your samples in columns of 8, left-to-right, top-to-bottom. Indicate the location of sample #1 on your plate (the plate itself as well as the seal on the plate) with an asterisk written in Sharpie pen. Sample 1 on your data sheet will be position A1 on your plate. Sample 2 on your data sheet will be position B1 on your plate. Sample 9 on your data sheet will be position A2 on your plate, and so on.

4. Empty wells in the middle of a plate and spoiled reactions will be charged.

5. Partial plates are accepted for 24 or more samples. If you have fewer than 24 samples, use 8-strip tubes, and trim the empties, etc.

Note: PCR plates inked with alpha-numerics are available at cost in my lab. They heat-seal very well and are standardized for my robotics. You can buy them for $50 for a box of 25. They are numbered and lettered along the margins, which is great when you (and I) are loading. Sign for them on a clipboard near my freezer. 8-strip tubes and caps can be ordered from many vendors. Check with me to see what they look like. Do not submit low-profile tubes or plates.

When and where do I drop-off my samples?

Drop off your samples in BIOSCI 119. Mornings are best. If the lab is locked, leave your samples in a nearby common room, and email me their location (Room 339, 1st freezer from the door, etc.)

Receiving Results:

What is the turn-around time to receive results?

Turn-around time varies a great deal from hours to 3 days, depending on how busy the lab is. Samples are run in multiples of 96, so if yours are the only 16 samples in the freezer, you will have to wait longer than if the lab is really busy.

How will I know when my sequencing results are completed?

Check your lab folder on bio-lemming to see if you have new data ready. You can check your folder any time; you do not need notification from me to do so.

  • If your samples are in my freezer by noon, wait 3 business days before making an inquiry to me, including the day you submit.
  • If your samples are in my freezer after noon, wait 4 business days before making an inquiry to me, including the day you submit.
  • After that time has elapsed, email me if you don't see your data. Remember not to count weekends or Duke University holidays.
  • It really helps move things along when your data sheet is in the dropoff folder before your samples get arrive in the freezer.

How can I view my results?

Most clients use Sequencher to view and edit their results from their lab data folders. However, two good free viewers are:

  • 4Peaks (designed for MAC OS X)
  • and FinchTV:.
  • Applied Biosystems has a free viewer.
  • Biology has a site-license for Sequencher. Check with your own departmental computer support to inquire about obtaining a copy.

Why did my samples give weak or no readable sequence?

I am happy to help you figure out what went wrong with your samples. Let me know MY run folder date which I assign to your returned data, so I can look up your data. If I can' t give you an answer, I will connect you with someone who can. I use a control on every run possible to test the performance of my instruments, and you should run your own standard with every submission, especially with large numbers of samples. An internal standard is most important if yours are the only samples on a run that fail. The University of Michigan DNA Core does a great job of explaining chromatogram interpretation and troubleshooting. Go to here and click on "Chromatogram Interpretation" or "Troubleshooting."

Will I get charged if my samples gave little or no readable sequence?

All samples are billed. If there is an instrument failure or a lab accident, the lab will reimburse you for the cost of the BigDye needed to rerun your reactions, and repeat electrophoresis will be free.

How can I get in touch with the sequencing facility?

Email (lisa.bukovnik@duke.edu) is the best way to get in touch with the sequencing facility. If I am not in the lab, and not on vacation, I will have access to email. My phone number is 919-660-7436, and I check messages compulsively. Mornings are the best time to drop by BioSci 119. Afternoons I am busy preparing samples. If I am not here (I have no staff at the moment) when you stop by, send me an email.

How do I get old results?

You are responsible for moving your data from the bio-lemming server to permanent media as soon as you receive it. I keep data backed up on DVD, so you can check with me in case of loss.

How will services be billed?

Services are billed monthly to the fund code you leave on your data sheet or sign-out sheets for BigDye or other services. For questions about your bill, email the lab accountant, Jennifer Foreman.