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The Genomics Core Facility provides a rapid, dependable, and economical service for DNA sequencing for the research community located in the Department of Biology at East Carolina University. We utilitze the 3130 Genetic Anyalyzer from Applied Biosystems and BigDyeTerminator v3.1 chemistry to produce quality sequence data of over 600 bp using investigator-provided templates from PCR, plasmid prep and other large DNA samples.


These samples can be run with primers the Facility has in stock, or with custom primers provided by the researcher. Quality of DNA samples is the key to good sequencing results. To ensure quality and timely return of results, of your results, please review our requirements for sample submission and guidelines for shipping samples.

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The versatility of the 3130 has allowed us to work with researchers to develop our newest service-analyzing fluorescently labelled primer extension products. Primer extension products, traditionally run with radioactive labels, can now be labeled with one of the five fluorescent colors recognized by the 3130 thereby saving time and health concerns. The five color capability of the ABI 3130 means that mutiplexing of samples with four fluorescent dyes is possible, with one color reserved for size standards.

We encourage you to use the links on the left side of the screen to learn more about our facility and the services we offer, or contact us directly.

Pricing

Sequences are $8.00 per single template/primer reaction for University researchers, and $10.00 per single template/primer reaction for non-University researchers. Primer Extension is $4.00 per sample for University members, and $6.00 for researchers outside the University. If the researcher has prepared the sequencing reactions, the cost for electrophoresis only is $1.50 per sample.

Turn-around time

Results will be ready in 2 – 3 working days. An email will be sent to the address(es) listed on the request form when the results are posted to the server.

Server access

A folder will be created for a researcher the first time he/she sends samples to the Genomics Core Facility. The PI will automatically have access to his/her folder on the server. To add access for students to their folder, the PI will have to send an email to Denise Mayer (mayerd@ecu.edu) to request access. Please incude the PirateID in the email.

Re-runs

DNA sequencing reactions may fail for various reasons, and the researcher may ask the Facility to re-run their samples. If it is determined the reaction failed due to poor template, or primer difficulties, the researcher will be charged for the re-run. If, however, it is determined the Facility is at fault, there will be no additional charge to the researcher.

We accept plasmid, PCR products, cosmids, phage, BAC, and bacterial genomic DNA samples. Your samples can be sequenced with either facility stock primers, custom primers sent with your order, or custom primers synthesized at the facility. Clients submitting custom primers should follow these general guidelines to ensure quality results. Clients who have large inserts can take advantage of our primer walking service to get information on their entire clone. Finally, for clients who prefer to set up their own reactions, we offer a "capillary run only" service. Please note that we can only accept capillary runs that have been set up using Applied Biosystems BigDye chemistries. Please specify in the "Comments" section of the OnCore System which chemistry has been used.

Sequencing samples are run on the Applied Biosystems 3130 DNA Genetic Analyzer. The AB 3130 uses a five-color dye system and provides up to 700 bases of usable sequence data per reaction.

Submit primers diluted to 5 uM (pmol/µl). Primers that don't have the concentration written on label will be assumed to be 5 uM.

Primer names are limited to eight characters. We reserve the right to shorten names exceeding 8 characters.

Do not use punctuation marks (except for a dash), Greek letters or symbols in the template or primer names. The software will not accept them.

We use 1 µl of primer (at 5 uM) per reaction. Please send extra to allow for evaporation during shipping and so that we may redo a reaction, if required, without having to contact you for additional primer.

We carry several of the commonly used primers in stock, available to researchers free of charge. At this time, we have the following primers:

Primer

Abbr.

Sequence 5' -> 3' 

pUC/M13 universal forward

for

CGC CAG GGT TTT CCC AGT CAC GAC 

pUM/M13 universal reverse 

rev 

TCA CAC AGG AAA CAG CTA TGA C 

T7

T7 

TAA TAC GAC TCA CTA TAG GG 

T3 

T3 

ATT AAC CCT CAC TAA AGG GA 

SP6 

SP6

ATT TAG GTG ACA CTA TAG 

-20 M13 forward

-20for 

GTA AAA CGA CGG CCA G 

Keep the Tm of custom primers between 54-58°C. If it is possible please do not use primers with a Tm below 50°C.

Check your primer for any dimers or hair-pin loops that may be formed. There are many software packages that can be used to check for structual problems.

Template Types

DNA samples commonly run at the Genomic Core Facility are PCR-generated products, plasmids, bacteriophage inserts or cosmids. Purity is the critical part of sequencing-if the templates are not of the utmost purity the sequence results will be disappointing. For plasmid preps, we find that Sigma, Quiagen and Promega kits provide consistant purity, yielding quality sequence data. For cleaning PCR products, we have had very good results using the High Pure PCR Product Purification kit from Roche, and also with ExoSAP-IT from USB Corpororation. Gel purification is also a good option. Please be aware ExoSAP-IT does not remove primers from the PCR product, just chews it up. This makes quantifying by spectrophotometry inaccurate. Quantification of the PCR product will have to be an estimation from running on a gel.

Important!! Do not use Tris/EDTA (TE) buffer to redissolve DNA or primers! EDTA chelates the Mg++ that is required for the sequencing reactions. Phenol from alkaline lysis mini preps is not tolerated at all in the sequencing reaction. Similarly, incomplete removal of cellular components such as RNA, proteins, polysaccharides, and chromosomal DNA will adversely affect your sequencing results. Residual ethanol in the template can also lead to a failed sequencing reaction. A thoroughly dried column prior to the elution of DNA is imperative.

Template Concentration and Volume

The table below shows the concentration needed of template needed. A picture of the agarose gel the template was run on is also required. Please note the volume of template used in the lanes on the gel.

Template Type

Concentration

Volume 

Plasmid DNA, 1 - 10Kb

200 ng/ul 

10 ul/reaction 

Plasmid DNA, 10 - 20Kb

400 ng/ul 

10 ul/reaction 

PCR Products, 100 - 500 bp

20-30 ng/ul 

10 ul/reaction 

PCR Products, 500 - 1000 bp

30 - 50 ng/ul 

10 ul/reaction 

Large DNA (lambda, BACs, PACs etc.)

200 ng/ul 

1 ug 

Bacterial Genomic DNA

500 ng/ul 

2 -3 ug 

Sample Information

Write names of templates and primers CLEARLY on top of the 1.5-ml tube exactly as it is on your sequencing order. Data will be posted with primer names, eg. templateA1_primer1.

If any of your samples (primers or templates) are going to be used in multiplereactions, please submit only one tube each containing enough volume for all of the reactions.

Template names are limited to eight characters. We reserve the right to shorten names exceeding 8 characters.

Do not use punctuation marks (except for a dash), Greek letters or symbols in the template or primer names. The software will not accept them.

If label tape is used, position it so that the tube will still fit into a microfuge. Please don't put Scotch tape over handwritten labels. If the tape comes off, so does anything written underneath it.

The volume we request is at least double the volume necessary for the concentration required. This saves time if the sample needs to be re-run. If the concentration is lower, more volume is required. If the concentration of your template is significantly lower, quality of the sequence results may also be lower.

To submit a sample or samples to the ECU Sequencing Facility, please print out the proper form and fill it out in its entirety. Please be sure to include the billing information-samples will not be processed without this information. For information on template and primer volumes and concentrations, please check our Sample Guidelines tab above.

For University members:

DNA sequencing samples are submitted using this form. For primer extension products use this form. Please put one template/primer pair per line. It is not necessary to ship your samples on wet or dry ice, just make sure they are packaged securely. Microfuge tubes sent in a 50 ml conical tube do very nicely. If you would like to bring your samples to us yourself, you can put them in the mailbox labeled "Denise Mayer" in N113 in the Howell Science Complex, otherwise send them through campus mail to:

Denise Mayer
Genomics Core Facility,
551 Biology

For Non-University Customers:

Sequence samples are submitted using this form. Please put one template/primer pair per line. Samples should be sent through an overnight service to the address below. It is not necessary to ship your samples on wet or dry ice.

Shipping Address:

East Carolina University
Department of Biology
Genomics Core Facility
Howell Science Complex
Mail Stop 551
Attn: Denise Mayer
Greenville, NC 27858

You will receive an email informing you when your results have been posted to the server for you to pick up. The directions on how to access the server are as follows:

To map a network drive using Windows:

  • Right click on "My Computer" and select "Map network drive" 
  • Choose a drive letter or you can just use the default letter. 
  • In the "folder" field, type in the path to your folder using the Pirate ID of the PI where indicated: \\cf1\coreprod\genomics\PI PirateID here 
  • Check the box that says "reconnect at logon" so this drive will automatically appear each time you log on to your computer. 
  •  Click finish -Enter your ECUID and password when prompted

On a Mac using the FTP program Fetch:

  • You can download the FTP transfer program Fetch. Be sure to download the instructions containing the serial number for the ECU site license. 
  • When you open the program it automatically goes to a new connection window. 
  • In the box next to "Hostname" type: core.ecu.edu 
  • Leave "Connect Using" set at FTP -Put in your ECUID and password where indicated 
  • In the box next to "Initial Folder" type: core1/docs/genomics/PI PirateID here
Files

For each sample sequenced, there will be two files - a text file with a .seq extension containing just the DNA sequence results, and an electropherogram file with a .ab1 extension. The text file can be opened in Word or any word processing program by launching the program, then using the File...Open command to bring up the Open File dialog box. Locate the drop-down box that allows you to change the file type the program will see. Be certain it says "All Files", instead of "Word Files" or whatever else it may be set on.

Software for Viewing Sequencing Electropherogram Files

The electropherogram file can be opened in Windows with Chromas from Technelysium or ChromaTool from Biotools, Inc. Sequence Scanner Software from ABI is also available. Mac users can download 4Peaks from mkentosj.com.

IMPORTANT: While our staff strives to generate the highest quality results by conducting routine quality assurance assessments each day, we do not edit or otherwise review your results. We encourage you to review your electropherograms and edit the sequence files according to your specific standards. If you are a new user and require help in editing and interpreting electropherograms please contact us.

Software for Viewing Fragment Analysis Files

The .fsa files for primer extension can be viewed in STRand or Peak Scanner from Applied Biosystems.

The newest service from the Genomics Core Facility is primer extension analysis. This is an electrophoresis-only service using researcher-generated primer extension products and LIZ size standards on our ABI 3130 Capillary Electrophoresis Genetic Analyzer. Since the standard is labeled with the fifth dye, users can genotype a greater number of markers in a given capillary, compared to the four-dye system.

Supported Fluorescent Labels

The ABI 3130, using Dye Set G5, will detect fragments labelled with the fluorescent dyes FAM (blue), VIC (green), NED (yellow) and PET (red). The fluorescent dye HEX may be used in place of VIC, and TAMARA may be used in place of NED.

Size Standards

Commonly the LIZ 500 standard is used, allowing for size determinations fom 50 - 500 base pairs.

The LIZ size standard, available from Applied Biosystems, is available in different kits ranging from 120 – 1200 base pairs.

In this chromatogram of the LIZ500, note the asterick above the 250 base pair standard peak. According to ABI, this peak is sensitive to small temperature variations on capillary electrophoresis instruments, and should not be used. Also, primer peaks may interfere with the proper detection of the 35 base pair standard. Be sure to take this information into consideration when optimizing and analyzing your results.

Optimization

It is the responsibility of the researcher to optimize the primer extension. A good way to this is to run serial dilutions of the primer extension product. In general, dilutions of 1:10 to 1:50 are necessary.

View Results

The data may be viewed in STRand or in Peak Scanner from ABI.

Genomics Core Facility
Department of Biology
Howell Science Complex N201
Mail Stop 551
Phone: 252-328-2607/252-328-6302
Fax: 252-328-4178
Greenville, NC 27858

Dr. Ed Stellwag
Director, Genomics Core Facility
East Carolina University
Department of Biology
252-328-6302
stellwage@ecu.edu

Denise Mayer
Research Operations Manager
East Carolina University
Department of Biology
252-328-2607
mayerd@ecu.edu

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