DNA Sequence Analysis
DNA Core Facilities provide competitive and timely DNA sequencing in an efficient and cost effective manner. We offer two Sequencing services, High Throughput $4/rxn and Standard $6/rxn for on-campus and $9 for off-campus users. The samples will be run on either our ABI 3130xl Genetic Analyzer or our ABI 3730xl DNA Analyzer.
Sample and Primer Submission
- Please fill in all of the information we need on a sequencing order form which is available at the DNA core lab or our web site.
- Template and primer should be dropped off in refrigerator near Room 5071 Sanger Hall.
- All templates and primers must be labeled and the concentration must be clearly written. Please limit your sample name to 8 and primer to 5 characters or less.
- A photograph of 1ul of sample on agarose gel is welcome.
- Put your order form in the box near the NARF entrance, Room 5-071B.
- Sample drop off hours: 9:00am-5:00pm, Monday-Friday
Purity and quality of DNA template play very important roles to obtain good results. The ABI 3130xl and 3730xl capillary sequencers are very sensitive to template and primer preparation. The templates need to be free of salt and other contaminants which can affect the quality of sequencing data.
We recommend using QIAGEN plasmid preparation kits or Promega Wizard mini-prep kits. Concentration should be determined by A260/280 OD. Submit 10ul of sample per sequencing reaction. Please see us if you have a big insert >3kb or high GC and AT rich template.
We still recommend using QIAGEN or Promega’s PCR Product clean-up kits to clean up your PCR Products. Submit 10ul of sample for sequencing reaction.
For high throughput sample $4/rxn: The user provides templates in 96 well plates. Core personnel perform the sequencing reaction and analysis data on the ABI 3730xl DNA Analyzer.
DNA template should be diluted in dH20 not TE buffer. Concentrations as below:
Please dilute your sample to the concentrations as listed. Due to time restrictions, a fee of $5.00 will be added per dilution.
The Core Guarantee: Control reactions are run with every sequencing reaction. Thus, there is no charge if the control reactions fail, and a 50% charge for each failed reaction.
We provide a free standard primer. Please see the list on the bottom of sequencing order form.
- M13F (-21), M13F (-47), M13R (-22)
- T7P Promoter, T7T Terminator
- SP6, T3
- KS and SK
Non-standard primers should be carefully quantified spectrophotometrically (preferably scanned 230-280 nm) and provided with the concentration 20ng/ul. Submit 10ul for each reaction.
A few "rules of thumb" for sequencing primers
- 18-25 bases in length
- 40-60% G+C, preferably with 3' G or C
- No homopolymeric tracks of more than 3 bases
- Minimize secondary and tertiary structure
DNA Sequence Analysis
|$4.00/rxn||96-well high-throughput sample for on- and off-campus user|
|$6.00/rxn||Standard sample for on-campus|
|$9.00/rxn||Standard sample for off-campus|
|$5.00/rxn||Repeating sample (if customer requests)|
|$3.00/rxn||Failed reaction (poor template or primer)|
If a system or sequencing reaction error has occurred,
your sample will be repeated at no cost to you.
Data Turn-Around Time
- 2-3 days
- If you drop off your sample before 9:00am your sample will be performed the same day. After 9:00am your sample analysys will be performed on the next business day.
For each of these services, you will be notified by e-mail when your samples have been processed. Data will be provided as electronic copy as ASCII sequence files or as raw data. There are several sites that offer PC and Macintosh freeware for viewing the electropherograms. Data can also be provided as hardcopy electropherograms at $1 per chromatogram.
NARF responsibilities are limited to providing the service of DNA sequencing. If for any reason DNA sequencing fails, we will assist the user in troubleshooting of the problem, and decide the best possible strategy to solve the problem. An extra fee might be charged for such advice if the problem is not due to an error generated by this core laboratory. We thank you for your understanding.