We utilize the 3130 Genetic Analyzer from Applied Biosystems and BigDyeTerminator v3.1 chemistry to produce quality sequence data of over 700 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.

Electropherogram

 Pricing

Sequences are $3.50 per single template/primer reaction for University researchers, and $4.00 per single template/primer reaction for non-University researchers. 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.

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.

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 consistent 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 Corporation. 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.

The table below shows the concentration needed of template needed. 

Template Type

Concentration

Amount in reaction

Plasmid DNA

75-200 ng/ul

200 ng

PCR Fragments

15-30 ng/ul    

30 ng

Large DNA

(lambda, BACs,

 PACs etc.)

200 ng/ul       

1 ug

Bacterial Genomic DNA

500ng/ul

2-3 ug

 

Sample Packaging & Labeling:

- 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, e.g. templateA1_primer1.

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

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

- Do not use punctuation marks (except for a dash or underscore), 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. 

-We request you send at least double the volume necessary for one reaction, in case the sample needs to be repeated.

 

Primers:

- 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.

* 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 rerun a reaction, if required, without having to contacting you for additional primer.

Our facility has available (at no charge to the client) the universal primers pUC19 (forward and reverse), -20 M13 forward, T3, T7 and SP6. The sequences to these primers can be found below. Clients can also provide their own primers. A volume of  5 ul of a 5 uM solution per reaction should be submitted. The following recommendations will help in designing optimal sequencing primers:

·             Primer length should be at least 18 bases long to be sure of good hybridization to the template, and longer primers will decrease the risk of binding to more than one site on the template.

·             Runs of 3 or more of a single nucleotide, especially guanine, should be avoided.

·             Primers with melting temperatures (Tm) above 45° C will produce better results.

·             The G-C content should be between 30-80%. If the G-C content is less than 50%, it may be necessary to design a primer longer than 18 bases to keep the Tm above 45°C.

·             Make sure the primers do not have a secondary structure, and cannot hybridize to form dimers.

pUC/M13 Forward: 5'-CGC CAG GGT TTT CCC AGT CAC GAC-3'

pUC/M13 Reverse: 5'-TCA CAC AGG AAA CAG CTA TGA C-3' 

pUC/M13 Forward(-20): 5’-GTA AAA CGA CGG CCAG-3'

T3: 5'-ATT AAC CCT CAC TAA AGG GA-3'

T7: 5'-TAA TAC GAC TCA CTA TAG GG-3'

SP6: 5’-ATT TAG GTG ACA CTA TAG-3’

 To submit a sample or samples to the Genomic Core 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 University members:

Please download and print out this form. Please put one template/primer pair per line. Samples can be sent through campus mail to the address below, just make sure they are packaged securely. Microfuge tubes sent in a 50 ml conical tube do very nicely. It is not necessary to ship your samples on wet or dry ice. A copy of the gel the samples were run on should be admitted as well to aid in determining sample purity and troubleshooting if necessary. 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, or deliver them to N203.

For Non-University Customers:

Please print out 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

 Genomics Core Facility

Department of Biology, MS 551

Howell Science Complex N203

Attn: Denise Mayer

Greenville, NC 27858

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