Home > Research Services > DNA Sequencing > DNA Sequencing FAQ
Customer Accounts
   Q: I am a new customer, how do I set up an account?

On our homepage (www.genewiz.com) you will see a "Register" link to set up a new account in the upper right-hand corner. Clicking on this link will allow you to set up your login name (your e-mail address) and password. This process takes only a few minutes; then you will receive an account activation e-mail. Simply click on the link in this e-mail to activate your account. You can now login to your account using your e-mail address and the password you just created. You are ready to submit your first order!


   Q: How can I try out your service?

We encourage you to try GENEWIZ services! As a new customer we offer a free trial coupon to try our DNA sequencing services. All you have to do is to send an email to dnaseq@genewiz.com or call our toll free number. Your Sales Executive will contact you and provide the necessary information.


   Q: What is the price of my order?

For DNA sequencing, price display during the order submission process is available for some accounts. If this is a feature you are interested in activating, just contact your GENEWIZ Sales Executive to find out if it is available for your institution. You can also contact your Sales Executive for a quotation. One more option is to contact GENEWIZ after you have submitted your order, to obtain the price for that specific order.


   Q: Can I open an account and submit samples if I live outside the US?

Absolutely! We welcome customers from all over the world. When you set up a new online account, you will be asked for your state. Living outside the USA, choose “other” from the drop down menu. Please provide the correct city and postal code. All of the other fields are filled out as designated.


   Q: Why should I choose GENEWIZ?

GENEWIZ is an operational excellence company with a customer-driven strategy. We have become the leader in DNA sequencing and molecular biology services, including gene synthesis, by providing the best combination of value - fast turnaround, specialized expertise, competitive prices, and friendly customer support. We continuously innovate to produce the best results with the greatest convenience for our customers.


DNA Sequencing Sample Submission
   Q: What is the difference between Pre-Mixed, Pre-Defined, and Custom?

Choose "Pre-Mixed" if you have adjusted your sample concentration following our guidelines and have already added your own sequencing primers, or are requesting one of GENEWIZ’s free universal primers.

Choose "Pre-Defined" if you have adjusted your sample concentration following our guidelines, but are supplying your primer in a separate tube for GENEWIZ to add (small additional charge may apply).

Choose "Custom" if you want GENEWIZ to adjust your sample concentration and add your primers (additional charge may apply).


   Q: How do I prepare my samples so I will get good DNA sequencing results?

While the majority of reactions sequence well, you should avoid several situations that can create unfavorable results. Here are guidelines to help you optimize sequencing results:

1.) Closely follow the DNA Sequencing Sample Submission Guidelines. A sub-optimal DNA-to-primer ratio is often the culprit for poor quality results.

2.) Make sure that your primer can adequately bind to your template. Insufficient primer binding can lead to disappointing results. Sequencing primers should be about 18-24 bases in length with a Tm of 56-60 degrees. The GC content should be about 45-55%, which is generally needed in order to get an 18-24 base primer in the Tm of 56-60.

3.) Avoid inhibiting contaminants. Salts, EDTA, alcohol, protein, RNA, detergents, cesium and phenol are some of the most common contaminants that can cause poor quality or failed reactions.

4.) Be sure to choose the correct protocol. Some templates (hairpins, GC-rich, etc.) benefit from a special protocol to ensure a good sequencing reaction. We offer alternative protocols at an additional cost that are highly effective in sequencing through difficult regions. These protocols can be requested from the “Special Requests” drop down menu on your online submission form.


   Q: What types of DNA templates can be sequenced?
   Q: How much DNA and primer should I submit?

Please closely follow the DNA Sequencing Sample Submission Guidelines.


   Q: How do I determine my DNA concentration?

We recommend that you check your DNA concentration and associated 260/280 and 260/230 ratios with a spectrophotometer such as a NanoDrop. You can also estimate your DNA concentration on an agarose gel by loading a small amount of DNA and running it with a marker of known concentration.


   Q: Can I use 8 strip tubes if I am submitting less than 8 samples?

Yes, you can just cut off the unused tubes and use them for your next submission, or send the entire 8-strip with labels on just the tubes containing your samples.


   Q: Can you sequence very short (100 bp) PCR?

We highly recommend that you sequence PCR products that are no shorter than 200 bp (500 bp or more is ideal).

GENEWIZ’s Molecular Biology team can also TA clone and sequence your PCR product. This strategy is particularly useful for shorter PCR products, and PCR products containing multiple sequences. TA cloning and sequencing can deliver your DNA sequencing results in as little as 3-5 business days. Please contact GENEWIZ Project Management for more information.


   Q: What universal primers do you have?

We provide these free universal primers:
http://www.genewiz.com/public/universalprimers.aspx


   Q: Can you help me if I my sequence is GC-rich and hard to amplify?

Yes, GENEWIZ has proprietary protocols that have proven successful in sequencing templates with high GC content, as well as templates with other prohibitive secondary structures such as hairpins. When filling out sequencing instructions, select one of our proprietary protocols listed in the "Special Protocol" column. Additional charges apply.


   Q: How long do you keep my samples?

We keep your DNA samples for one week, unless otherwise specified. If you need either your DNA or your primers for future orders, please specify this in the “Comments” section of your order. Just write “Please keep my DNA, primer, or both for future orders.” We can store your DNA for one month or your primers for up to a year.


   Q: How do I know if my vector has a binding site for a universal primer?

Check your vector map or manufacturer’s protocol. It will tell you what primer site is present or what primer is recommended for sequencing. You can then compare this to the GENEWIZ universal primers. Simply click on the “GENEWIZ Free Universal Primer” link and you will see the free universal primers supplied by GENEWIZ with their respective sequence information.


   Q: What do I need to provide for you to perform PCR cleanup on my samples?

Submit your unpurified PCR products with your primer(s) in separate, well-labeled tubes. Also include a gel image and the amount loaded on the gel. Be sure to select “Custom” as your service type and choose “PCR clean up” from the Special Request column when submitting your order. We will perform the clean up and then optimize the sequencing reaction based on your gel image.


   Q: What type of 96-well plate should I use to submit my samples in bulk format?

Feel free to use any brand of PCR plates and caps. Be sure to load your plates in the column or vertical direction. Here are a few suggestions of plate/cap combinations that work well for a tight seal:

Nunc (through Fisher): 96-Well PCR Plates- 12-565-536, 12-566-133, or 12-566-134, Caps- 12-565-810 or 12-575-123

Nunc (through VWR): 96-Well PCR Plates- 73520-694, 83009-280, or 73520-696, Caps - 73521-364 or 73521-066

USA scientific- Plates- 1402-9400, 1402-9600, 1402-9700 with caps- 1400-0800 or 1400-3800

ABI- Plates 4306737, Caps 4323032


   Q: How should I label my 8-strip tubes?

Label your tube on the SIDE with the tube ID generated from your online order form. The tube ID is made up of your initials and the tube number. See figure below (If GENEWIZ was submitting samples, the tube ID would be GW01-GW08-as labeled below):


DNA Sequencing Results
   Q: When will I receive my DNA sequencing results?

If you requested Same Day service, you will have your results within 7 hours of samples arriving at GENEWIZ (when samples are received by 10 AM). If you requested Standard Service, you will have your results by noon the next business day following sample receipt at GENEWIZ. Note that customers using our San Diego, Boston and Maryland Labs enjoy the Faster Local Service. Please contact us for details and eligibility. We always send an e-mail notification when your results are available. Feel free to login to your account at any time to check the status of your order.


   Q: What software can I use to view my results?

From the “Recent Results” section of your account, you have access to the page where you can download your data (both the .seq and .ab1 files). The .seq file is a text file and can be opened with any program that can view text files (Wordpad, Notepad, etc.) If you are having difficulty opening these files, you can change the file extension from .seq to .txt as a last resort to view it.

The .ab1 file is your chromatogram, and requires chromatogram viewing software to view these files. Please visit www.genewiz.com/tools.aspx (or click on the “Tools for Viewing Sequencing Data” link on the menu) for a number of free software programs that are available for viewing trace or chromatogram files.


   Q: How long do my results stay in the online system?

We strongly recommend that you download all of your results and back them up on your laboratory computer. Our current policy is to store your results for 6 months (please note that any data older than 3 months will require transfer back from our archive and can take up to 24 hours after initiating the transfer request from your results page). This policy is subject to change at anytime, but changes, if any, will be clearly communicated.


About Primers
   Q: How do I design primers for sequencing?

We recommend designing your sequencing primers in a region that is 100 bases upstream of your sequence of interest. If you do not have the luxury of having this buffer, the closest you want the primer to be to your area of interest is 50-60 bases. Anything closer and you risk missing a portion of your area of interest. The primers should be about 18-24 bases in length with a Tm of 56-60 degrees. The GC content should be about 45-55%. Many vendors that provide oligo synthesis services have software into which you can plug your primer sequence to check for Tm, GC content and homodimerization. For the oligo purity, desalting is all that is needed for sequencing.


   Q: Can you design and order primers for me?

Yes, we can design and order primers for you; an additional charge applies. If you already have the primer sequence, you will simply fill out the “Sequencing Primer” order form. If you would like GENEWIZ to design the primer for you, please send the reference sequence with your area of interest highlighted or otherwise indicated to dnaseq@genewiz.com.


Troubleshooting
   Q: What should I do if my sequencing results failed?

If you are experiencing any difficulties with your order we are always happy to assist your troubleshooting efforts; all the troubleshooting that we perform is free of charge. Please call Technical Support at GENEWIZ or email us at dnaseq@genewiz.com for assistance.


   Q: What does it mean when I have a poly A/T region resulting in poor quality
       sequence?

When the polymerase encounters a homopolymeric region such as poly A or poly T in the sequence, it often results in subsequent non-specific or poor quality sequence due to slippage of the enzyme. When this happens, we generally recommend sequencing from the opposite direction to obtain the missing sequence data.


   Q: How can I ensure successful sequencing results for GC-rich PCR products?

In our experience, performing a PCR amplification using CleanAmp™ 7-deaza-dGTP Mix from TriLink BioTechnologies, improves amplicon yield and quality for targets that have GC content of 60% or greater. When using these amplicons with Sanger sequencing, you can receive longer sequence read length and improved Phred scores.


For more information about this product, please click here.


Additional information is also available in the white paper “Robust PCR amplification of GC-rich targets with Hot Start 7-deaza-dGTP.”


   Q: I have a very long primer and am getting bad results, why?

A longer primer has a higher Tm, which will not anneal to your DNA template properly during the sequencing reaction and would generate unsatisfying results. We recommend that you use our Universal Primers or design your own primers that are about 18-24 bases in length with a Tm of 56-60 degrees.


   Q: PCR primers were used for sequencing, why didn’t they work?
       Could the problem be a degraded primer?

While degraded primer could be the culprit, another possibility is that your PCR primers are good for PCR reactions but not suitable for sequencing reactions. For sequencing primers, we recommend that they are about 18-24 bases in length with a Tm of 56-60 degrees. Longer PCR primers usually do not generate good results for sequencing reactions. Also, not all PCR primers work well as sequencing primers. If your PCR primer is not generating good sequencing results, we recommend that you design a nested primer to use as your sequencing primer.


GxP/CLIA DNA Sequencing Services
   Q: Are you GLP (Good Laboratory Practice) or cGMP compliant?

GENEWIZ’s Quality System is developed in accordance with applicable FDA regulations described in Title 21, Part 58 of the Code of Federal Regulations for Good Laboratory Practice. In addition, our quality system includes elements of cGMP compliance (21CFR211, 600, ICH Q7A) to support biopharmaceutical manufacturing Quality Control testing. We offer GLP services including GLP DNA sequencing and GLP level molecular biology projects. For more details, please contact us at 877-GENEWIZ (436-3949) or Regulatory@genewiz.com.