DNA Sequencing - Frequently Asked Questions

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Samples

Q: How should I prepare the samples?

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

Non-Premixed Requirements (Per Reaction)
Sample Type Concentration Volume
Purified PCR product < 1kb 10-20 ng/μl 8 μl
Purified PCR product > 1kb 20-50 ng/μl 8 μl
Unpurified PCR product* N/A 8 μl
ExoSAP-it treated PCR product* N/A 5 μl
Plasmid <6kb 50-150 ng/μl 8 μl
Plasmid >6kb 100-200 ng/μl 8 μl
Primer 5 μM 5 μl

 *If you are sending ExoSAP-it treated or unpurified PCR products, the concentrations are not required. Please DO NOT dilute the samples before submission.

Mail-in customers: To save on shipping costs and time, please provide double the volume listed above in the event a repeat is needed.

Additional Guidelines:

  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. It is preferred that 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 a large number of samples in the following situations. We also recommend that you submit a control template and primers to facilitate troubleshooting should any problems arise:

    • You suspect a problem with your template
    • You are using a new template preparation method
    • You are using an untested primer
  1. It is important that the ration of a 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.
  2. 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.
  3. For PCR product, remove or disable any unincorporated dinucleotides.
  4. 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.

Q: Am I able to send in premixed samples? If so, how should I prepare them?

 We prefer samples to be sent in with the DNA and primers in separate tubes.  This allows us to check the concentrations individually and add the amount of DNA and primers appropriately according to our protocols. If you still prefer to premix samples, please contact support@etonbio.com for instructions. (Note: We are unable to troubleshoot and run free repeats for reactions with premixed samples.)

Primers

Q: What is the primer storage policy?

All orders will have primers stored for 2 weeks before they are discarded. 

You can request to save primers for a longer period when placing your sequencing order. Simply select "To Be Saved" in the Primer Source Column on the Sequencing Order Form.

Primers can be stored for free for up to one year:

    - All original primers, synthesized in house or provided by customer, will be stored for 1 year

    - All primer aliquots will be discarded after 3 months of non-use 

Customers will not be notified when primers are running low or are discarded.

If an order is placed with a stored primer that has been discarded the customer will be notified within 24 hours so they may submit or order a new primer for their order.

You can email support@etonbio.com before placing an order to check on the availability of your stored primer. 

 

Q: Where do I get primers?

There are 3 options:

  1. We provide many commonly used primers, free of charge. Please note that the primer sites on your samples may not be completely complementary to these standard primers. Therefore, check carefully before using these primers. Please click here to view the list of primers that Eton provides.
  2. You provide your own primer.
  3. Order a specialty primer through us. Follow the steps on the sequencing order form to place your order in one step or you can use our oligo ordering system separately to place your order.

Q: How should I prepare the primers?

If you provide your own primers, please follow these recommendations:

  1. Please provide at least 5 uM of primer per reaction.
  2. Select and design a primer that only primes at one location on your template and does not form secondary structures or primer dimers.
  3. Ideal primer length for sequencing is between 18 and 24 bp.
  4. Ideal Tm should be 60C, as we can not modify the annealing temperature on our PCR machines for individual orders.
  5. Primers should have about 50% G/C content.

Q: What amount of primer is needed?

Please provide us with at least 5 ul per reaction of primer
at concentration between 5µM to 10 µM.

Q: What primers does Eton Bio offer for free?

Please visit the Universal Primer page to view all free primers. If there is a standard primer you don't see on our list, please email support@etonbio.com with a request to add it to our list.

 

Q: Can I have my sequencing primer delivered?

All primers ordered when using the Sequencing order form will be stored in the lab for future use.  If you'd like your primer delivered you must place your order for oligo synthesis using our Oligo ordering form. Once the primer has been delivered, you will then need to make an aliquot to submit with your sequencing order. 

Please see Oligo FAQ for delivery and turnaround times.

How to Place an Order and How Samples are Picked Up

Q: How do I place an order and schedule a pick up?

If you are a new customer, you will begin by registering an account on our website.  Login to your account and click "Place Order" for the service you will be using. There are many options to help you fill in your reaction information. For a step-by-step explanation, please click "Download Order Instructions" on the first page of the order process.  Please make sure to include the ready time, whether the samples are Plasmid or PCR (if PCR, please list the purification method), sample size and whether they are premixed with primer. If you have any further information on the samples that you feel is beneficial for us to know, such as the samples are GC rich, please include that in the Special Instructions.

You will receive a confirmation email notifying you that the order was placed successfully.

Once we receive the order in our system, we will schedule a pick up for you same day, as long as it is placed before the cut off time (please see next FAQ).

Q: What is the sample pickup schedule?

For local pick up times or to see if you qualify for sample pick up, please contact your respective branch. Click here for contact information.

Q: Does Eton Bio take mail-in orders?

Yes. To prevent sample leakage, please make sure to cap your tubes tightly, preferably with parafilm.  If you are submitting a 96 well plate, please make sure the plate is sealed tightly with PCR strip tube caps. Then please ship your samples to the nearest branch. Click Here for branch locations.

Results

Q: How soon can I get the results?

If your sequencing order is placed before the morning cut off times or as Express Service, results will be available same day.  For regular sequencing orders, placed before the afternoon cut off time, results will be available before noon on the next day. Orders that are shipped to our branches will be processed the morning of their arrival and results will be available before 6pm.

Sequencing Orders that include synthesized primers as well as provided primers will be processed all together when the synthesized primers are ready.  If you need the data from the reactions with provided primers sooner, please order them separately from the reactions with the synthesized primers.

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.

For more specific information, please contact our Sales Team at sales@etonbio.com or 800-758-1630.

Q: How do I get the results?

We will send you an email when your results are ready.  The email with either contain the files as an attachment or a direct download link, depending on your personal choice while ordering. In order to directly download, choose "File FTP" when placing an order.  You will be able to access downloads from your order history on your account.

You will receive a zip file containing two files per sequence;  a chromatogram of the sequence (.ab1) and a text file (.seq). The zip files are compatible with both MAC and Windows.

Q: How do I view the results?

The text sequence file can be read with any available text editors such as Notepad or Microsoft Word. To read the chromatogram files, you will need to download a proper viewer.

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 the individual chromatogram file or file folder.

Q: What is your repeat policy?

If you were not satisfied with your results, please contact our customer service department by email (support@etonbio.com) or phone (800-758-1630) and they will be happy to troubleshoot your order. Upon review of the data, they can set up one-time repeats free of charge for failed reactions or reactions with unsatisfactory quality.

The original reaction will still be charged. In other words, we charge once and only once for both the original and repeat reactions.

Concentration requirements must be met in order to qualify for free repeats.

Q: Why did the repeats work when nothing changed with the samples or primer?

Although there is always a chance of human or machine error with sequencing, many times we do change the conditions for a repeat reaction. When a repeat reaction is requested, our customer service department looks over the original data and adjusts the amount of DNA and/or primer added or adds DMSO to help with the reaction. These simple adjustments can benefit the reaction and help to generate a much cleaner sequence.

Other

Q: How does Eton Bio price services?

We provide quotes per request.  Please email sales@etonbio.com for more information on pricing.

Q: Why should I use services from Eton Bio?

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.



Contact Support

If you have any questions not covered in this section, please feel free to contact us at 800.758.1630.

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Eton Bioscience, Inc.
5820 Oberlin Drive, Suite 108
San Diego, CA 92121
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