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
Considerations for Standard Sequencing
|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|
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|
|PCR fragment||5 µM|
|Bacterial Genomic||15 µM|
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|
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.
Dr. John Tine
Karen Preston, M.S.