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.

Template Concentrations

Primer Concentrations

B. Standard Primers (provided by CFG)

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.