Answers to FAQ:

Recommendations on Preparation of Samples:

We recommend the following in the preparation of your samples for successful DNA sequencing:

1. Make sure that the sample you submit only contains DNA and ddH2O, and is free of
   salts, EDTA, alcohol, protein, RNA, detergents, cesium and phenol.

2. Make sure that you have sufficient DNA in your sample for sequencing.

3. Preferably you have a unique DNA product in your sample. If you have a plasmid
    product, make sure to remove all bacterial genomic DNA.

4. We suggest you submit only a few samples to test out your conditions prior to
    submitting large number of samples in the following situations:
        1) You suspect a problem with your template;
        2) You are using new template preparation method;
        3) You are using untested primers.
    In the above situations, we also recommend that you submit a control template and
    primers to facilitate troubleshooting should any problems arise.

5. It is important that the ratio of 260/280 of your sample's O.D. reading is between 1.8
    and 2.0. Otherwise, there is a good chance that your reaction will fail or have a poor
    quality due to contamination or improper concentration.

6. We highly recommend that you run your templates on agarose gel before submission.
    There should be one, clearly defined band on the gel representing a particular
    template. Please make sure to have appropriate gel percentage, length of time and
    voltage.

7. For PCR product, remove or disable any unincorporated dinucleotides.

8. Certain sequence motifs and secondary structures may prevent high-quality results. In
    such situations, we will work with you to formulate a particular sequencing strategy
    to get the best possible results for you.

How much template is needed:

Concentrations of samples:

      PCR products:                                  10-20 ng/µl
      Plasmid:                                             100 ng/µl

So, we need about 2 to 3 µl of PCR products or 3µl of plasmid at above concentrations to meet the following requirement for one sequencing.

For successful sequencing, the following template amount is needed:

       PCR Products:          
             100-2000 bp                        10-40 ng
             >2000 bp                           40-100 ng

       Single Stranded Plasmid:                 100 ng
       Double Stranded Plasmid:                250 ng

If it is possible, we recommend doubling of the above amount in case we need to repeat the sequencing reaction in certain situations.

How to prepare premix samples:

If you would like to premix templates and primers, please follow these recommendations:
1) Please provide at least 8 µl of total volume per reaction;
2) In this 8 µl, please add 250 ng of DNA for plasmid, or 40 ng of DNA for PCR products;
3) In this 8 µl, please add 5 pmol of primer.
4) Please leave a note in Special Instruction to indicate your samples are premixed.

Examples:
-- If plasmid concentration is 100 ng/µl, and primer concentration is 5 µM, you need to
    add 2.5 µl of plasmid, 1 µl of primer and 4.5µl of dH2O to make the final volume 8 µl.
-- If the concentration is 20 ng/µl for PCR products, you need to add 2µl of DNA, 1 µl of
    primer and 5µl of dH2O to make the final volume 8 µl.

Where to get primers?

Three sources:
Source 1: We provide the following commonly used primers free of charge. Please note               that the primer sites on your samples may not be completely complementary
              to the following standard primers. Therefore, check carefully before using
              these primers.
              Please click here to view a list of primers that Eton provides.
Source 2: You provide your own primer.

How to prepare primers and how much primers are needed:

If you provide your own primers, please follow these recommendations:
1) Please provide at least 10 pmols primer per reaction;
2) Select and design primer that only primes at one location on your template and does
    not form secondary structures;
3) Ideal primer length for sequencing is between 18 and 24 bp;
4) Primers should have about 50% G/C content;
5) Primers should not form primer dimers or prime multiple times within your sample.

How to schedule a pickup and place an order?

You may login your account, click on "Place Order". Then, fill in the order form and
    sample information. No further actions are required on your part. All orders placed by
    4:00pm will be picked up on the same day for our San Diego branch. For our
    North Carolina branch, all orders placed by 3:00pm will be picked up on the same day.

What is the sample pickup schedule?

Normally we pickup samples between 2-4pm each working day for the San Diego branch. For orders that have more than 20 samples, we can pickup when they are ready before 4pm. For the North Carolina branch, we normally pickup samples between 2-3pm each working day.

Do you take mail-in orders?

Yes. Please make sure to cap your tubes tightly, preferably with parafilm. Then,
please ship your samples to:
          Eton Bioscience Inc.
          5820 Oberlin Drive, Suite 100,
          San Diego, CA 92121

The United States Postal Service does not deliver directly to our office in North Carolina. For orders sent to our RTP branch, use FedEx or UPS and ship your samples to:
          Eton Bioscience, Inc.,
          104 T.W. Alexander Drive
          Bldg 7
          Research Triangle Park, NC 27709

How soon can I get the results?

Generally, results will be available before noon on the next day if your samples are ready for pickup by 4:00pm for our San Diego branch or 3:00pm for our North Carolina branch.

For BAC/Phage sequenecing orders, we provide results within 48 hours.

For sequencing orders in combination with other service items, such as primer synthesis, sample purification, or primer walking etc., it may take a longer time depending on the specific services requested.

How do I get results?

There are two ways:

We will email you the results as soon as they become available;

We send zipped files for both Windows and Mac users; We will send .sit files for Mac users on demand. You may use WinZip or Stuffit to decompress the files. Top

How to view the sequencing results?

The text sequence file can be read with any available text editors. You may download proper viewers to read chromatogram files.

For Sequencher users, to make the results viewable, you will have to download the compressed file and decompress it. Then, start a New Project in Sequencher, and Import individual chromatogram file or file folder.

What is your repeat policy?

We will provide one-time repeat free of charge, if requested by scientists, for failed reactions or reactions with unsatisfactory quality, regardless of the cause of the failure or low quality. However, the original reaction will be charged. In other words, we charge once and only once for both the original and the repeat reactions.

How do you price your service?

Please go to pricing.

Why should I use the services from Eton?

We deliver services, not words. We are confident that our services will speak for themselves as time goes on. We understand that you have many choices. We hope that when making your choices, you will take the following factors into consideration: competitive pricing, efficiency and convenience, together with the willingness to improve continuously.

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