1. Standard (Single Reaction) Sequencing
A standard single reaction consists of one plasmid, PCR fragment, or BAC DNA template sequenced with one primer to produce one chromatogram (A single template sequenced with both forward and reverse primers would comprise two reactions, for example). This option is best when your template/primer combinations are below the threshold for discounted economy sequencing. For common plasmid vectors we provide a number of standard sequencing primers free-of-charge (see list below) or customers can provide custom-designed primers. Guidelines for sequencing are below.
2. Bacterial Genomic (BG) Sequencing
DNA sequence can be obtained directly from bacterial chromosomal DNA by Sanger sequencing, and we offer this as an option. Larger genomes cannot be sequenced directly by this technology. Because of reagent requirements, BG reactions are more costly than standard single reactions. Customers should recognize that this application is at the technical limits of the Sanger sequencing technology, and thus results are inconsistent. As such, even when templates of apparent quality and sufficient concentration are submitted, good results are not always achieved. Because of this, success with BG templates is not guaranteed.
3. Economy Full Plate Sequencing
If you have large numbers of samples to be sequenced, we offer discounted pricing for submission of samples in full 96-well plates for sequencing. To obtain this pricing, customers must provide templates and primers already loaded into a 96-well plate with the following guidelines:
- A minimum of 80 samples (template + primer) are required.
- An Applied Biosystems 96-well plate (part no. N801-0560) must be used for samples. Plates from other manufacturers are not compatible with our instrument.
- Primers should be diluted to a concentration of 5μM, with 1 μl added per well.
- Templates should be added in appropriate quantities (see Template Concentrations chart below), with a total well volume including template, water, and primer of 5 μl/well.
- Plates should be sealed with an adhesive foil or plastic sheet cover, and labeled on the side. If you mark the top surface of the plate as a pipetting guide, mark lightly with a black, ultrafine pen. Avoid colored inks; these can fluoresce and interfere with the detection of sequencing products.
Contact us before setting up and submitting your first plate to inquire about the status of the capillary array. Occasionally, a capillary will become non-functional, in which case the corresponding well on the plate should not be used.
Note: Economy sequencing is not available for BG DNA templates.
|Standard (1 template/1 primer)||$10.00|
|Bacterial Genomic DNA (1 template/1 primer)||$14.00|
|Economy Full Plate, per well||$3.00 ($288.00/plate)|
Please, contact us about pricing for Non-Academic institutions
To Request Sequencing Services
Guidelines for Submission of Samples for DNA Sequencing
A. Template and Primer Concentrations
Recommended concentrations for templates and primers are shown below. At these concentrations we use 1 µl template and 1 µl primer per reaction. If you cannot achieve the stated template concentration, we can compensate by adding more template volume (up to 6 µl) per reaction, but note that we have observed a decline in sequencing quality with DNAs less than 1/4 the recommended concentration, regardless of this adjustment. Submission of primers that are not at the requested concentration may result in a delay in the processing of your samples.
Please submit templates/primers separately in 1.5 or 0.5 ml microtubes. Please label to match exactly what you write in columns of the submission sheet. Include enough volume for all requested reactions plus some “extra” volume. Please submit a minimum of at least 5 µl even if only 1 µl will be used. For shipping, wrap tops of tubes with Parafilm.
|Plasmid||100 - 300 ng/µL|
|Bacterial Genomic||3 µg/µL|
|PCR frag. 100 - 200 bp||3 ng/µL|
|PCR frag. 200 - 500 bp||10 ng/µL|
|PCR frag. 500 - 1,000 bp||20 ng/µL|
|PCR frag. 1,000 - 2,000 bp||40 ng/µL|
|PCR frag. > 2,000 bp||100 ng/µL|
|Template Type||Primer Concentration|
|PCR fragment||5 µM|
|Bacterial Genomic||15 µM|
B. Standard Primers (provided by CFG)
|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|
C. 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 (methanol, isopropanol, etc).
Templates submitted should be accurately quantified by determining both A260 and the A260/A280 ratio (ratio should be at least 1.7). Submitted templates should be resuspended or eluted in water or 10 mM Tris/HCl pH 7.5-8.0, not TE, as EDTA will inhibit the activity of the sequencing enzyme. An agarose gel run is also useful to rule out genomic bacterial DNA contamination and degraded plasmid DNA. PCR fragments must appear on a gel as a clean single band of the expected size.
The CFG recommends isolation of plasmid/BAC/BG templates by use of an appropriate commercially available spin-column type kit (i.e., Qiagen, Promega, etc.). For plasmids, miniprep-scale kits are perfectly acceptable. Old-fashioned methods like “rapid boiling minipreps” or phenol-chloroform extractions should not be used as the remaining contaminants can damage the capillary array.
PCR products must be purified to remove residual primers/nucleotides. For PCR products, we recommend purification with commercial kits (i.e., Qiagen, Promega, etc.). With any kit, pay attention to the instructions for avoiding carry-over of ethanol or excessive salt in the eluted or resuspended product. Note: We do not recommend gel-purification of PCR products unless absolutely necessary. Clean-up of PCR products with products like Exo-SAP can be acceptable, but it makes the accurate determination of template concentration difficult. In such cases, the risk of having an over- or under-loaded reaction is borne by the customer.
D. Quality Control
We take significant steps to verify the quality of 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. We split every sequencing reaction in two and load in two separate capillaries so we can detect these rare occurrences. 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 below. Please note that for Economy Full-Plate Sequencing, reactions are NOT split and thus this double-check is not available.
E. Policy on Repeat Reactions
Because of the quality control measures described above, it is usually possible for us to determine by examining the raw data and instrument run logs whether the sequencing reaction failure is due to 1) our reaction set-up or instrument failure, or 2) the quality/quantity/appropriateness of the DNA template and/or primers provided by the customer. We will automatically repeat any reaction (without charge) if it appears that the failure is type 1. (We recommend that you ship enough of your samples to allow us to repeat your reactions.) However, most sequencing failures in the industry are type 2, which is outside the control of the sequencing facility. If you feel your failed or low quality results are unexpected, please contact us for a discussion of possible remedies. If it appears to be a matter of inaccurate DNA quantification by the submitting lab (e.g. chromatogram peaks too low or too high [off-scale]), on request we can do a few repeat reactions with altered template volumes (assuming enough is supplied) to see if that improves results. If large numbers of samples need to be repeated due to inaccurate DNA quantification by the submitting lab, we will have to charge accordingly. Accurate quantification is the responsibility of the laboratory submitting the samples. If we need to re-quantify DNAs by Nanodrop spectrophotometry, we assess a charge of $1.50 per sample.
For mystery failures, where DNA quantification does not seem to be the issue, we can do a few repeats with the same templates/primers if requested. However, if they fail a second time, we will charge for the repeat reactions as well as the originals. If they succeed when repeated, we will only charge once. Please note that sequencing bacterial genomic DNA by Sanger technology is a process at the technical limits of the application, and even when template of sufficient concentration is submitted, good results are not always achieved. Therefore we cannot guarantee success with this type of template.
Please let us know if you are submitting “difficult templates” such as those containing inverted repeats, high GC content, homopolymeric runs, etc. We can alter reaction conditions to increase the chance of success with such templates, but extensive secondary structures sometimes require re-sequencing with different primers (i.e. ones that bind on the other strand) to complete the sequence.
Guy Russo, M.S.