DNA Sequencing - Frequently Asked Questions
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Q: How should I prepare the samples?
We recommend the following in the preparation of your samples for successful DNA sequencing:
- DNA Amount Needed:
Concentration: Between 75 - 125 ng/µl
Amount: At least 600 ng in 6 µl diH2O per reaction.
- Purified PCR products:
Concentration: between 5 - 30 ng/µl, unless you order purification from Eton
*no concentration requirement for EXOSap purified PCR products.
Amount: At least 60 ng in 6 µl diH2O per reaction.
- 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.
- Make sure that you have sufficient DNA in your sample for sequencing.
- 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.
- 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:
- You suspect a problem with your template
- You are using a new template preparation method
- 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.
- 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 y our reaction will fail or have a poor quality due to contamination or improper concentration.
- 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.
- For PCR product, remove or disable any unincorporated dinucleotides.
- 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: What amount of the sample is needed?
|Sample Requirements: The following guidelines need to be met to qualify for free repeats|
|Concentration: between 75 - 125 ng/µl. |
Amount: at least 600 ng in 6µl diH2O per reaction.
|Purified PCR products:|
|Concentration: between 5 - 30 ng/µl, unless you order purification from Eton |
*no concentration requirement for EXOSap purified PCR products.
Amount: at least 60 ng in 6µl diH2O per reaction.
Q: How should I prepare premixed samples?
If you would like to premix templates and primers, please follow these recommendations:
- Please provide at least 8 µl of the total volume per reaction.
- In this 8 µl, please add 250 ng of DNA for plasmid, or 40 ng of DNA for PCR products.
- In this 8 µl, please add 5 pmol of primer.
- Please leave a note in the area marked Special Instructions to indicate your samples are premixed.
-- If plasmid concentration is 100 ng/µl, and a primer concentration is 50 ng/µl, you need to add 2.5 µl of plasmid, 1 µl of primer and 4.5 µl of dH2O to make the final volume 8 µl.
--If the concentration is 20 ng/µl for PCR products, you need to add 2 µl of DNA, 1 µl of primer and 5 µl of dH2O to make the final volume 8 µl.
Q: Where do I get primers?
There are 2 options:
- 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.
- You provide your own primer.
Q: How should I prepare the primers?
If you provide your own primers, please follow these recommendations:
- Please provide at least 10 pmols primer per reaction.
- Select and design primer that only primes at one location on your template and does not form secondary structures.
- Ideal primer length for sequencing is between 18 and 24 bp.
- Ideal annealing temperature should be 60C, as we can not modify the annealing temperature on our PCR machines for individual orders.
- Primers should have about 50% G/C content.
- Primers should not form primer dimers or prime multiple times within your sample.
Q: What amount of primer is needed?
Please provide us with at least 2ul per reaction of primer
at concentration between 30ng/ul to 70ng/ul.
Q: What primers does Eton Bio offer for free?
To view the primers that Eton provides, please click here.
How to Place an Order and How Samples are Picked Up
Q: How do I schedule a pickup and place an order?
Login to your account and click "Place Order." Fill in the order form and sample information. The order form includes an easier way to upload your data from an excel file and a 96 well plate feature, which you can find under the "Order Form" section. Please make sure to include the sample size, whether the samples are premixed with primer and whether the samples are Plasmid or PCR products (If PCR, please indicate whether they are purified, not purified or EXOSAP-it treated). 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. 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 the San Diego branch, our cut off time for pick up is 4pm (for local companies/universities), but we typically pickup samples between 2-4pm each working day from local companies/universities. For companies/universities in the Los Angeles area, our cut off time for pick up is 3pm. The sample pick up cut off time varies for other companies/universities outside these areas, please contact us at, 858-558-1630, for specific times. For the North Carolina and Boston branches, the cut off time is 3pm and we typically pickup the samples between 2-3pm each working day. Please make sure your order is placed before these times to make sure the samples will be picked up same day.
Q: Does Eton Bio take mail-in orders?
Yes. Please make sure to cap your tubes tightly, preferably with parafilm. Then please ship your samples to:
5820 Oberlin Drive, Suite 108
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:
104 T.W. Alexander Drive
Research Triangle Park, NC 27709
Q: 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 4:00 pm for our San Diego branch or 3:00 pm for our North Carolina and Boston branches.
Results can be received by email or by direct download. In order to directly download, choose download when placing an order. A download link will be sent to the email address provided. You will be able to access downloads from your order history on your account.
For BAC/Phage sequencing 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.
Q: How do I get the results?
There are two ways:
- We will email you the results as soon as they become available.
- We send two files, a zip file with a chromatogram of the sequence and a text file. 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. 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 the individual chromatogram file or file folder.
Q: What is your repeat policy?
We will provide a 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 of low quality. However, the original reaction will be charged. In other words, we charge once and only one for both the original and repeat reactions.
If you were not satisfied with your results, please contact our customer service department by email (firstname.lastname@example.org) or phone (858-558-1630) and they will be happy to troubleshoot your order and set up repeats for you.
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.
Q: How does Eton Bio price services?
Please go to 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.
If you have any questions not covered in this section, please feel free to contact us at 800.758.1630.
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