The DNA sequencing facility at CFG offers DNA sequencing services for a wide range of templates including plasmid DNA, PCR fragments, cosmid DNA, bacterial genomic DNA, BAC and Phage DNA.
We perform sequencing reactions using Applied Biosystems BigDye terminator chemistry. Reactions are run on ABI Prism 3730XL capillary-based DNA sequencers.
Please, contact us about pricing for Non-Academic institutions
|1 template/1 primer||$10.00|
To Request Sequencing Services
- For first time users, complete and submit a Customer Information Form that you can download directly in either word or acrobat formats.
- Complete and submit a Request for DNA sequencing Form that you can download directly in either word or acrobat formats. Please submit additional forms if you are submitting more than 10 samples.
- Arrange to send template sample(s) to the CFG. Please see Template Concentrations below for appropriate amounts of template to submit.
- CFG provides standard sequencing primers (see below) at no charge. If using your own primers, please see Primer Concentrations below and include with template shipment.
- Policy on
Template Concentration PCR fragment Concentration Plasmid 100 - 300 ng/µL 100 - 200 bp 3 ng/µL Cosmid 1 µg/µL 200 - 500 bp 10 ng/µL Bac 1 µg/µL 500 - 1,000 bp 20 ng/µL Bacterial Genomic 3 µg/µL 1,000 - 2,000 bp 40 ng/µL Phage 1 µg/µL > 2,000 bp 100 ng/µL
The concentration of primers that we require depends on the type of template to be sequenced. Please follow the guidelines below. We use 1 µl of primer per reaction. Please provide enough primer so that reactions can be repeated, if necessary. Submission of primers that are not at the requested concentration may result in a delay in the processing of your samples.
Template Type Primer Concentration Plasmid 5 µM PCR fragment 5 µM Cosmid 10 µM Bac 10 µM Phage 10 µM Bacterial Genomic 15 µM
Standard Primers (provided by CFG)
Primer Sequence Length (bases) M13 Forward 5'- GTA-AAA-CGA-CGG-CCA-GT-3' 17 M13 Reverse 5'- AAC-AGC-TAT-GAC-CAT-G-3' 16 M13/pUC Forward 5'- CCC-AGT-CAC-GAC-GTT-GTA-AAA-C-3' 23 M13/pUC Reverse 5'- AGC-GGA-TAA-CAA-TTT-CAC-ACA-GG-3' 23 T3 5'- ATT-AAC-CCT-CAC-TAA-AG-3' 17 T7 5'- AAT-ACG-ACT-CAC-TAT-AG-3' 17 SK 5'- TCT-AGA-ACT-AGT-GGA-TC-3' 17 KS 5'- CGA-GGT-CGA-CGG-TAT-CG-3' 17 SP6 5'- ATT-TAG-GTG-ACA-CTA-TAG-3' 18
Recommendations for Template Preparation
For successful cycle sequencing, it is crucial that templates be of high purity and appropriate concentration. Possible deleterious contaminants include bacterial proteins and carbohydrates, inorganic salts and organic solvents such as methanol, isopropanol, etc.
Templates submitted should be accurately quantitated by determining both A260 and the A260/A280 ratio (ratio must be at least 1.8). Submitted templates should be resuspended in water or 10 mM Tris/HCl pH 7.5-8.0, not TE, as EDTA will inhibit the activity of the Taq enzyme. An agarose gel run is also useful to rule out genomic bacterial DNA contamination and degraded plasmid preparation.
The CFG recommends isolation of plasmid templates by use of one of the commercially available kits (i.e., Qiagen, Promega, etc.). PCR products must be purified to remove residual primers. For PCR products CFG recommends isolation with commercial kits (i.e., Qiagen, Promega, etc.). Because these systems use salts and ethanol, extensive washing of pellets to remove salts and complete drying to remove ethanol is required.
We take significant steps to quality control each sequencing run in our facility. We include a sequencing standard (consisting of a standard plasmid sequenced with a standard primer) in each run. If we do not obtain good quality sequence from this standard, it is indication that there may have been a general problem with the reactions, and thus any failed reactions in such a run would be repeated at no charge to the investigator.
Rarely, we find improper loading of a capillary or some other failure of a specific capillary. To compensate for this, every sequencing reaction is split in two and loaded in two separate capillaries. Thus, in the event of an isolated problem with a capillary we can obtain data from the other member of the sample pair.
If both members of the pair fail, it is unlikely that a loading problem, etc, has occurred with both capillaries. We would then proceed with our resequencing policy as stated above.
Policy on Repeat Reactions
As a general rule, we will repeat failed reactions one time, but we can repeat no more than six reactions per order. If these reactions are successful, the remaining reactions of the order will be repeated. The customer will be charged only for the first set of reactions. If the second reactions fail, the order will be closed and the customer will be billed for the first set of reactions only.
If, when evaluating data from initial failed reactions, it appears to us that an adjustment of DNA concentration may result in a successful reaction, we will do so (again, up to six reactions). If the reactions are successful, we will re-do the remaining reactions and bill as above (i.e., once for each reaction). If the second reactions still fail, the order will be closed and the customer will be billed for the first set of reactions only. A new sequencing request must be submitted for any further work to be performed on templates in orders that have been closed.
If we have consistent reaction failures over multiple orders from a customer, we will contact the customer and attempt to resolve any problems before proceeding with additional resequencing.
Investigators can either provide purified templates (plasmids, PCR fragments, genomic DNA, Phage DNA, Cosmid DNA.) or provide material from which CFG staff will purify the templates (bacterial cultures, templates and primers for PCR, etc.). If providing templates, please review our recommendations for template preparation (see link above) to insure highest quality results.
Standard primers (see link above) are provided at no charge by CFG. Custom primers can be synthesized at the CFG or provided by the investigator. In general, a single run should result in at least 500-700 bases of high quality sequence information.
Turn-around time is generally 1-2 days from receipt of the template and primers, depending on the workload at the facility. Additional services, such as template preparation, will add to the turn-around time. The investigator will receive the results as a sequence file and a color electropherogram for each template/primer combination. Files will be sent by e-mail to all investigators. We can also send files by mail or fax if required.