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Northwestern University Feinberg School of Medicine
Center for Genetic Medicine
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Sanger Sequencing

NUSeq offers traditional sequencing services through a partnership with ACGT. This partnership provides the following benefits to NU researchers:

  • Industry-leading data quality
  • Preferred pricing for Northwestern (lower than historical in-house prices)
  • Next business day turnaround time
  • Expert technical support
  • Campus sample drop-off, with courier pickup and sample shipping at no costs to users
  • Centralized ordering and billing in NUcore

 

Common Reasons for Failure of Sequencing Reactions

Please review THIS pdf for troubleshooting your samples. This pdf was prepared by ACGT, Inc and should help you with all of the common issues. If you are still having issues please contact ACGT. 

 

Sanger Sequencing Pricing

Please log in to NUcore to see the preferred ACGT pricing for Northwestern University users. Please note that external users are not covered by this partnership and should order from ACGT directly.

Order Sanger Sequencing

 Sample Preparation

Plasmid and purified PCR DNA samples are accepted templates. Please refer to the ACGT site for sample submission requirements. Samples may be submitted in individual tubes, strip tubes, or 96-well plates. Samples submitted in tubes receive the same low rate as those submitted in 96-well plates. Samples submitted in 96-well plates are considered as High-Throughput and may take additional time to process.

ACGT provides a list of universal primers free of charge. When using these universal primers, please provide their name(s) as exactly listed on the ACGT site (copy and paste works well). Any deviation, e.g., name change (T7 to T7Promoter, or CMV Forward to CMV) or addition of extra space or underscore, will cause the system to identify it as a user supplied custom primer. As a result universal primer will not be added leading to sample failure. If an LCO service requires universal primer(s), please don't mix your own primer with your template. In case you prefer to add your own universal primer please reflect this in the primer name, e.g. T7-added.

User-supplied custom primers need to be submitted following the ACGT Sample Submission Requirements linked above.

 Order Submission

Sample orders are placed in NUcore. Please contact us if you need to set up a NUcore account. You don't need to set up an account with ACGT to submit orders. The first time you order Sanger sequencing through NUcore, an account will be automatically created for you at ACGT using your email address as the username. Your sample information will be transmitted to the ACGT server from NUcore automatically. At the end of the ordering process in NUcore, an order number is generated. Please make a note of this number, as it is required for sample drop-off.

Available Services

There are three options to choose from:

  • Low Cost: template and primer premixed
  • Standard: template and primer submitted separately, concentrations to be provided by user
  • Premium: template and primer submitted separately, concentrations to be random checked and then generalized by ACGT

Difficult-to-Sequence Templates

For samples that have high GC content and/or hairpin structure, please mark them on the order form accordingly so that the right sequencing protocol will be used. There is no additional charge for complex templates.

Submitting Samples in 96-Well Plates

Samples may be submitted in individual tubes, strip tubes, or 96-well plates. If you need to submit samples in 96-well plate format, sample information can be provided in bulk via an Excel CSV file upload (choose ">Upload File" at the top of the sample order form). For manual plate sample input, please use the "Submit as a plate" option above the sample form. When plating samples please arrange them by rows (or columns) in a continuous fashion to avoid problems with sample info entry.

Notes for using the bulk CSV file upload option:

  • Samples should be sequential, without gaps.
  • Any lines left blank in the file will appear as blank lines in the web form, where they must be deleted before the form can be saved.
  • If you delete lines between samples in the file they will still appear as blank lines in the form.
  • You MAY delete blank lines after the last sample in the file and these lines will not appear in the form.
  • For the columns labeled "Hairpin (Y/N)" and "High GC (Y/N)", a blank is equivalent to "N" (i.e. the box will be UN-checked on the form).

Only one plate should be uploaded to each form. If you upload a second file it will over-write the existing form contents.

To submit multiple plates, enter the number of plates on the home page of the core when ordering and NUcore will create an item in the cart for each plate. NOTE that each item in the cart will have a unique order number. Each plate of samples MUST be labeled with the correct order number.

 Sample Drop-Off

Once you have placed a Sanger sequencing order in NUcore, you may drop your samples off to a nearby dropbox. The locations of dropboxes are:

Evanston Campus:

Cook Hall, Room 4161 (2220 Campus Dr., Evanston, IL 60208)

Chicago Campus:

NUSeq Core, Tarry Building, Room 2-760 (300 E. Superior St, Chicago, IL (60611) 

Robert H Lurie Medical Research Center, 7th Floor by the stairs (303 E Superior, St. Chicago, IL,60611)

Sample bags are provided at the NUSeq Core location to facilitate sample submission. Please note that your samples MUST be labeled with your NUcore order number. The order number can be marked on the sample bag, or you may insert a printed copy of the order receipt into the bag.

Daily sample drop-off cutoff time is 2 p.m. for Evanston campus and 3 p.m. for Chicago campus.

 Getting Results

Samples will be processed and results generated in the next business day (large number of samples submitted in 96-well plates may take additional time to process). Once results become available, you will receive a notification email from ACGT. Simply follow instructions in the email to download the data in a zipped file.

The zipped file contains electropherograms (*.ab1) and sequences (*.ab1.seq). The electropherograms can be viewed using programs such as FinchTV, which is freely downloadable from Geospiza (works on major operating systems including Windows, Mac OS, and Linux). The sequence file (*.ab1.seq) is in text format and can be opened with any text editing software such as Notepad. 

 Sample Disposal and Storage

Samples are stored for two weeks post-sequencing then discarded, or banked at ACGT upon request at no charge.

 Sample Troubleshooting

In case of sample failure or unsatisfactory results, please contact ACGT Tech Support (dnaseq@acgtinc.com or toll-free at 800-557-2248) for troubleshooting options.

Frequently Asked Questions

 How do I set up an account?

The NUSeq Core Facility utilizes NUCore as an ordering platform. All core users must have a NUCore account to order any service. If you already have a NUCore account, click here to log in.

 How do I submit my full-service or ready-to-run sequencing order?

All sequencing samples are submitted through our online NUCore system.

 What do I do if I can't submit the online form?

If you are unable to log in to NUCore or cannot submit the form for any reason, please email nuseq@northwestern.edu.

 What do I do if I made a mistake on the sequencing submission form?

Sequencing forms cannot be edited once they are submitted. If any changes need to be made to the order, you need to cancel it and resubmit it. Only new orders can be canceled. You cannot cancel an order once we have begun processing it.

To cancel an order:

  1. Log in to NUCore
  2. Click on the Order Name for the order you wish to cancel
  3. Click the Delete Order button under Order Details at the top of the screen

 Can I still submit my traditional sequencing order in paper form?

No. Traditional sequencing orders must be submitted via our online NUCore system, as it is tied to billing and sending results.

 How do I prepare my samples for sequencing (template concentration, primer concentration, etc.)?

Plasmid and purified PCR DNA samples are accepted templates. Please refer to the ACGT site for sample submission requirements. Samples may be submitted in individual tubes, strip tubes, or 96-well plates. Samples submitted in tubes receive the same low rate as those submitted in 96-well plates. Samples submitted in 96-well plates are considered as High-Throughput and may take additional time to process.

ACGT provides a list of universal primers free of charge. User-supplied custom primers need to be submitted following the ACGT Sample Submission Requirements linked above.

 What universal primers does the core have for sequencing?

ACGT provides a list of universal primers free of charge. User-supplied custom primers need to be submitted following the ACGT Sample Submission Requirements linked above.

 When can I expect my results?

Samples will be processed and results generated in the next business day (large number of samples submitted in 96-well plates may take additional time to process).

 How do I view my results?

Once results become available, you will receive a notification email from ACGT. Simply follow instructions in the email to download the data in a zipped file.

The zipped file contains electropherograms (*.ab1) and sequences (*.ab1.seq). The electropherograms can be viewed using programs such as FinchTV. The sequence file (*.ab1.seq) is in text format and can be opened with any text editing software such as Notepad.

 What is the core’s policy on troubleshooting failed reactions?

In case of sample failure or unsatisfactory results, please contact ACGT Tech Support (dnaseq@acgtinc.com or toll-free at 800-557-2248) for troubleshooting options.
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