Roy J. Carver
Biotechnology Center

Submission Guidelines for the Core DNA Sequencing Facility at the University of Illinois, Urbana-Champaign

About Our Services

The Core Facility provides Sanger DNA sequencing and fragment analysis services to researchers at domestic or international academic institutions, government agencies and private companies. We are open from 8:30am to 5:00pm Monday-Friday with the exception of campus holidays. Core Facility personnel can be reached by phone at 217-265-6814, or by e-mail at dna-seq@illinois.edu. The laboratory is located in Room 334 of the Edward R. Madigan Laboratory on the University of Illinois campus. If you plan to ship samples to our facility, please use the following address:

DNA Core Sequencing Facility
1201 W. Gregory Drive, 334 ERML
Urbana, IL 61801

 

If you are an off-campus user, please contact us to set up payment before you submit samples.  We cannot process any samples without a payment agreement in place.  Please email dna-seq@illinois.edu for assistance.

All sequencing and fragment analysis samples are analyzed on Applied Biosystems 3730xl automated sequencers with 50cm capillary arrays. These instruments are extremely fast and accurate. The typical turnaround time is 2 business days from the date of submission. All orders are completed on a first-come, first-serve basis, regardless of affiliation.

The Core Facility handles both low-throughput (1-95 samples per order) and medium-throughput (full 96-well plate per order) projects.

 

Sample Handling

Samples for low-throughput sequencing and fragment analysis orders (1-95 samples) must be submitted in clear or light-colored 1.5mL tubes with attached lids (snap caps). Do not submit samples in strip tubes, 0.6mL PCR tubes, or in tubes that are darkly colored or that have screw caps. Clearly print the template or primer name in blue or black permanent marker on the top of the tube. Clearly print the user name and date of submission on the side of the tube. Template and primer names must be identical to the CoreLIMS request form and not more than 10 characters long. Please do not label tubes with tape or stickers. If you are shipping tubes to our facility, you may want to use a little parafilm to secure the lids and prevent leakage.

Samples for medium-throughput sequencing and fragment analysis orders (96 samples) must be submitted in clear 96-well PCR plates. The plates must be sealed before shipping to prevent evaporation or cross-contamination. Please seal plates with tightly fitting strip caps, or a high-quality adhesive tape such as BioRad Microseal 'B' Adhesive Seals (#MSB-1001). On the skirt of the plate, clearly print the CoreLIMS order number, the user last name and the date of submission.

 

How To Submit Templates and Primers for DNA Sequencing

For Sanger DNA Sequencing, we can accept purified PCR products, plasmid and large DNA constructs such as cosmids, fosmids and BACS.

 

Template Purity

PCR products must be purified to remove residual primers, dNTPs and reagents from the amplification reaction. If multiple bands are amplified, a gel purification kit must be used to extract the target band.

Plasmid and large construct templates must be free of genomic DNA, RNA, salts (including EDTA), proteins and organic solvents. Most commercially available kits are sufficient for purifying any type of DNA for sequencing; Applied Biosystems has also published an alkaline-lysis/PEG precipitation procedure that adequately purifies DNA and is relatively inexpensive. Regardless of the method, samples must be eluted in water or EB. Please do not elute purified DNA in EDTA as the presence of salt will inhibit the cycle sequencing reaction.

 

Template Concentration and Volume

Inaccurate DNA concentration is the #1 reason that DNA sequencing reactions fail. To obtain consistent, high quality sequencing results we recommend quantifying DNA templates via a 1% agarose gel using a mass ladder (companies like NE BioLabs carry several kinds). This method allows you to verify the size of your target DNA, as well as visualize contaminants such as RNA, genomic DNA and/or non-specific amplification products.

Quantification may also be done via spectrophotometer or fluorometer, but this method can be less accurate depending on the instrument that is used and how carefully it is calibrated. If using a spec, the A260/A280 ratio should be within the range of 1.7-2.0. A lower ratio is indicative of protein or phenol contamination; a higher ratio is indicative of excessive RNA contamination. Our facility uses and can recommend the Qubit® Fluorometer (Invitrogen). Many customers have reported problems when using the NanoDrop™ for one reason or another; we advise you to be very cautious of any readings obtained from this instrument and to consider using a secondary (or altogether different) method of quantification.

Please submit all DNA templates at the following concentrations:

Type	Required Concentration	Required Volume per Reaction*
PCR: 100-200bp	10-20 ng/L	5.0 L
PCR: 200-500bp	20-30 ng/μL	5.0 μL
PCR: 500bp-1kb	30-50 ng/μL	5.0 μL
PCR: 1kB+	50-60 ng/μL	5.0 μL
Plasmids	100-200 ng/μL	5.0 μL
Cosmids, fosmids and BACs	250-300 ng/μL	5.0 μL

* If a single DNA template will be sequenced with multiple primers, we recommend combining the separate volumes into a single tube.

 

Sequencing Primers

The Core Facility stocks 20 standard primers that can be used to sequence a variety of templates. When choosing a standard primer, please consult your vector sequence or map to determine whether or not our primer sequences are an exact match with your template. Unlike PCR, DNA sequencing reactions do not work well if there are single base pair mismatches between the template and the primer.

Primer Name	Sequence 5'-3'	Tm	Notes
BGH-rev	TAG AAG GCA CAG TCG AGG	58°
CMV-for	CGC AAA TGG GCG GTA GGC GTG	80°
DuetUP1	GGA TCT CGA CGC TCT CCC T	66°	For forward sequencing of MCS1 in DUET vectors
DuetDOWN1	GAT TAT GCG GCC GTG TAC AA	68°	For reverse sequencing of MCS1 in DUET vectors
DuetUP2	TTG TAC ACG GCC GCA TAA TC	68°	For forward sequencing of MCS2 in DUET vectors
EGFP-C	CAT GGT CCT GCT GGA GTT CGT G	72°
EGFP-N	CGT CGC CGT CCA GCT CGA CCA G	81°
M13For-21	GTA AAA CGA CGG CCA GT	58°
M13For-40	GTT TTC CCA GTC ACG AC	54°
M13Rev-24	AAC AGC TAT GAC CAT G	45°
M13Rev-48	AGC GGA TAA CAA TTT CAC ACA GGA	70°
SP6-pro	GCT ATT TAG GTG ACA CTA TAG	52°
T3-pro	ATT AAC CCT CAC TAA AG	50°
T7-pro	TAA TAC GAC TCA CTA TAG GG	56°
T7-term	GCT AGT TAT TGC TCA GCG G	61°
pET-Upstream	ATG CGT CCG GCG TAG A	63°	For forward sequencing of MCS1 in DUET vectors only; NOT for use with regular pET vectors
pGEX5'	GGG CTG GCA AGC CAC GTT TGG TG	82°
pGEX3'	CCG GGA GCT GCA TGT GTC AGA GG	78°
pQE-For	CCC GAA AAG TGC CAC CTG	66°
pQE-Rev	GTT CTG AGG TCA TTA CTG G	54°

Custom primers can also be used to sequence DNA templates. Custom primers do not need to be OPC or HPLC purified for use in DNA sequencing reactions. Custom primers should be submitted at a concentration of 5.0-10.0 pmols/μL and a volume of 5.0 μL per reaction. Please do not combine primers with the templates - submit them in separate tubes. If a single primer will be used to sequence multiple templates, we recommend combining the separate volumes into a single tube.

 

Instructions for submitting a DNA Sequencing order on the CoreLIMS site

1. If you are a member of one of the University of Illinois campuses, please create a login account, then login and immediately enter a payment option via the "Payment Manager" link on the main menu. For University of Illinois users, only a C-FOP (FOAPAL) number can be used for payment.

   If you are an off-campus user, please contact us to set up payment before you submit samples.  We cannot process any samples without a payment agreement in place.  Please email dna-seq@illinois.edu for assistance.

2. On the main menu, select the Sequencing Order Form hyperlink.
    
3. Choose the Sequencing, Ready-to-Load with Clean-Up or Ready-to-Load service option depending on the type of sample you are submitting. Enter the number of requests you'd like to create for this order and hit SUBMIT. Note: A request is defined as 1 template combined with 1 primer, and the maximum number of requests that can be in a single order is 96.
    
   • The Sequencing option is for purified DNA templates that have not yet been run in a BigDye reaction.
   • The Ready-to-Load with Clean-Up option is for completed BigDye reactions that need purification before electrophoresis.
   • The Ready-to-Load option is for completed BigDye reactions that are purified and need only electrophoresis.
      
4. On the next webpage, select a payment option from the drop-down menu at the top. If you need to add/modify/delete a payment option, please do it at this time, as you will not be able to modify it later without losing the information entered into the table below.
    
5. If you are using the Ready-to-Load with Clean-Up or Ready-to-Load option, enter the reaction names into the first column of the table. In the second column, select a DNA type from the drop-down menu and provide the vector name if prompted (not applicable to PCR samples). Use the "Fill" button at the top of each column to populate all rows at once with the information entered in the first line. Below the table, indicate whether you'd like the chromatograms edited (up to 800bp, or to the end of the PCR product if smaller) to remove ambiguous base calls. Type in any additional notes into the Comments field and then hit submit to enter the order. Print off 2 copies of the final order summary that appears and bring (or mail) one of them with your samples. Keep the other copy for your records.
    
6. If you are using the Sequencing option, enter the template names into the first column and the template concentrations (in ng/μl) into the second column. In the third column, enter the total size of the template in kilobases (i.e., if your PCR product is 800 base pairs, enter it as ".800"). In the fourth column, select a DNA type from the drop-down menu and provide the vector name if necessary. In the Primer Name column, choose one of our 20 standard primers from the drop-down menu or select the "custom" option at the bottom of the list. If you choose a standard primer, the primer concentration field will be automatically filled in. If you are using a custom primer, please enter the concentration (in pmol/μl) into the last column. Use the "Fill" button at the top of each column to populate all rows at once with the information entered in the first line. Below the table, indicate whether you'd like the chromatograms edited (up to 800bp or to the end of the PCR product, if smaller) to remove ambiguous base calls, and whether you need to have any of the samples primer walked. Type in any additional notes into the Comments field and then hit submit to enter the order. Print off 2 copies of the final order summary that appears and bring (or mail) one of them with your samples. Keep the other copy for your records.
    
7. When your reactions are completed, you'll receive an automated e-mail directing you to log into the CoreLIMS website. On the main menu under the Core DNA Sequencing header, click on the "Data Retrieval" link. Select your order number from the list and hit submit. The next page will contain a table of data files for your order. To download results for an individual sample, right-click on the links next to each sample ("trace" is the chromatogram; "seq" is the text file). To download the whole order at once, click on the "3730 sequence" or "3730 trace" button at the top of the page, then right-click on the "Download File" link that appears on the next page. The site will then download a compressed (.zip) file of your data that can be extracted. Free software programs for viewing, editing and assembling DNA sequences are available through the "Software and Support Tools" link on the CoreLIMS main menu.

 

How To Submit Samples for Fragment Analysis

For our Fragment Analysis service, we accept unpurified (unless otherwise directed) PCR products generated by microsatellite, AFLP, RFLP, TRFLP, ARISA or other protocols. Our capillary instruments can analyze fluorescently-labeled fragments up to 1200bp in length, and they provide high run-to-run consistency with low background noise. Due to the limitations of our capillary electrophoresis platform, we cannot successfully perform temperature-dependent analyses such as DGGE or SSCP.

Size Standards

There are several size standards available that are labeled with either ROX (for up to 3-dye multiplexing) or LIZ (for up to 4-dye multiplexing) fluorophores. Your choice of size standard will be based on both the expected sizes of your fragments and how many dyes you want to multiplex. We stock all size standards here at the facility and they are included in the analysis price. Here is a complete list of our standards:

•  LIZ120 - analyzes fragments that are 50-120bp in length with up to 4-dye multiplexing
•  ROX400 - analyzes fragments that are 50-400bp in length with up to 3-dye multiplexing
•  ROX500 - analyzes fragments that are 50-500bp in length with up to 3-dye multiplexing
•  LIZ500 - analyzes fragments that are 50-500bp in length with up to 4-dye multiplexing
•  LIZ600 - analyzes fragments that are 50-600bp in length with up to 4-dye multiplexing
•  ROX1000 - analyzes fragments that are 50-1000bp in length with up to 3-dye multiplexing
•  LIZ1200 - analyzes fragments that are 35-1200bp in length with up to 4-dye multiplexing

Fluorescent Dye for Primers

Once you have selected a size standard, use the chart below to determine which fluorophores are acceptable for labeling your primers. These are the only dye combinations that are approved by Applied Biosystems for the 3730xl. For questions about alternative fluorophores, please contact lab personnel.

Size Standard	Compatible Fluorophores
LIZ120	dR110 dR6G dTAMRA dROX
ROX400, ROX500, ROX1000	6-FAM HEX NED
LIZ500, LIZ600, LIZ1200	6-FAM VIC  NED PET

Sample concentration, purity and volume

Fluorescence on the 3730xl is relative; that is, the intensity (peak height) of an individual amplicon is affected by its own concentration and the concentrations of other amplicons in the sample. To help determine optimal DNA concentrations, we offer one free test plate to each new user. We recommend that you select up to 12 samples that represent a variety of marker sets for your project and perform serial dilutions ranging from 1/2 to 1/64. Load 5.0 μL of each undiluted sample in the top row, followed by 5.0 μL of each dilution in successive rows. Please leave row "H" open on the test plate for the controls. Use the following chart as a guide:

 

 

As stated earlier, sample purification is not necessary unless the samples have high salt concentrations that will interfere with the electrokinetic injection on the 3730xl. The results of the dilution test plate will help us determine if a purification step is required. The required minimum volume for each sample is 5.0 μL. Directions for tubes and plates can be found in the "Sample Handling" section above.

Instructions for submitting a Fragment Analysis order on the CoreLIMS site

1. If you are a member of one of the University of Illinois campuses, please create a login account, then login and immediately enter a payment option via the "Payment Manager" link on the main menu. For University of Illinois users, only a C-FOP (FOAPAL) number can be used for payment.

   If you are an off-campus user, please contact us to set up payment before you submit samples.  We cannot process any samples without a payment agreement in place.  Please email dna-seq@illinois.edu for assistance.

2. On the main menu, select the Fragment Analysis Order Form hyperlink.
3. Enter the number of requests you'd like to create for this order and hit SUBMIT. Note: A request is defined as 1 template combined with 1 primer, and the maximum number of requests that can be in a single order is 96.
4. On the next webpage, select a payment option from the drop-down menu at the top. If you need to add/modify/delete a payment option, please do it at this time, as you will not be able to modify it later without losing the information entered into the table below.
5. Select the appropriate size standard for your project from the drop down menu. 
6. Enter the sample names into the first column of the table. In the second column, select a sample type from the drop-down menu. Next, select the dyes that you've used to label your primers. Use the "Fill" button at the top of each column to populate all rows at once with the information entered in the first line. Below the table, type in any additional comments and then hit submit to enter the order. Print off 2 copies of the order summary that appears and bring (or mail) one of them with your samples. Keep the other copy for your records.
7. When the analysis is completed, you'll receive an automated e-mail directing you to log into the CoreLIMS website. On the main menu under the Core Fragment Analysis header, click on the "Data Retrieval" link. Select your order number from the list and hit submit. The next page will contain a table of data files for your order. To download results for an individual sample, right-click on the "seq" link next to each sample. To download the whole order at once, click on the "3730 sequence" button at the top of the page, then right-click on the "Download File" link that appears on the next page. The site will then download a compressed (.zip) file of your data that can be extracted.

 

Software Programs for Fragment Analysis Data

The data files generated by the 3703xl (.fsa) can be analyzed with GeneMapper (Applied Biosystems), PeakScanner (Applied Biosystems), or GeneMarker (SoftGenetics). We provide our customers with free access to two shared copies of the GeneMapper software via a remote connection to a computer at our facility. To obtain access to the shared copy, please complete the online GeneMapper Access Form. Your account will be set up as soon as possible and you will receive step-by-step instructions on how to connect to the remote desktop from your personal computer. A user manual for GeneMapper can be downloaded from the Core Fragment Analysis section on the main menu.
 

 

If you have questions or need more information,

please contact Core staff at 217-265-6814 or dna-seq@illinois.edu.