DNA Sequencing
Our DNA sequencing facility uses an ABI 3130 Genetic Analyzer to process DNA sequencing samples. We are located in room C110 in the C wing of the Microbiology building on the Colorado State University campus. See the Sample Submission page for detailed information.
There are three current service options available to customers: Premium, Standard and Economy.
First time users and customers having quality issues should read the Troubleshooting tips.
The premium level is designed for sequencing of large templates that require more Big Dye chemistry than normal sized templates, such as BAC’s, Cosmids, and Lambda DNA. Please contact David regarding genomic sequencing. It is also intended for customers who would like the core to perform the plasmid extraction from a colony or post PCR clean up.
- For BAC, Cosmid, Lambda please provide at least 1μg of DNA at 0.2 μg/μl.
- Customer supplied primer for large templates must be at 10 μM or 10 pmol/μl.
For customers who wish to have us perform the plasmid extraction you must follow these instructions: Prick an isolated colony with a pipette tip and put the tip in a 1.7 ml centrifuge tube. From this sample we can perform a reaction that uses theta replication and allows for sequencing with a minimal amount of cells. - For PCR clean up please provide 30 μl of sample. Gel electrophoresis must be done to verify amplification; however the core will perform the PCR clean up and quantify DNA.
- Customer supplied primers for sequencing should be at 3.2 μM.
In the event of an unsuccessful reaction we can provide troubleshooting suggestions. In addition, if there is sufficient sample remaining a rerun may be requested. Please do not submit new DNA for reanalysis.
Standard Level
The standard level is designed to replicate the current level of sequencing service. The customer must bring the plasmid or PCR DNA at the described concentrations. Any template not at these concentrations will have to be diluted and will be subject to additional charges.
- Plasmid DNA: 0.5 ug per reaction @ 50-100ng per µl. (Multiple samples must be at the same concentration).
- PCR DNA less than 1000 bases in length; 10 µl of DNA @ 10ng/µl concentration.
- PCR DNA more than 1000 bases in length; 10 µl of DNA @ 20ng/µl conc.
- Customer Supplied Primer: 5 ul's per reaction @ 3.2 µM or 3.2 pmol/µl.
Customers may request a rerun of your sample if sufficient DNA is provided, however, requisitions will be granted at Core's discretion. The amount of DNA described above provides us with enough DNA to perform a reanalysis.
Economy Level
This service is designed for customers who do sequencing regularly, and are confident that their reactions will work. The customer must bring both the template and primer mixed together at the specified concentration stated below. Please note that core supplied primers are not available on this service.
- Plasmid DNA:
Customer must combine 400 ng of plasmid template with 10 picomoles of primer in 24 µl total. - PCR DNA:
Customer must supply 2 ng of PCR DNA per 100 bases in length and 10 picomoles of primer in 24 µl total.
The following is an example of an acceptable PCR DNA setup:
| A customer has determined the concentration of their PCR template to be 20ng/ul with a length of 950 base pairs. Their primer has a concentration of 10 uM. First, determine how much DNA to add to the tube first. | |
| DNA needed = 2ng * ( PCR Size / 100 ) | |
| DNA needed = 2ng * ( 950 / 100 ) | |
| DNA needed = 19 ng | |
| Second, determine the amount of ul's needed to achieve 19 ng of DNA. | |
| Xul needed = (DNA needed) / (PCR Concentration) | |
| Xul needed = (19 ng) / (20 ng/ul) | |
| Xul of PCR DNA needed is = 1.0 ul | |
| Third, determine the amount of primer needed. | |
| 10 uM = 10 pmoles/ul | |
| X ul of primer needed = (10 pmol) / (primer concentration) | |
| Xul = (10pmol) / (10 pmol/ul) | |
| X = 1ul | |
| Last, combine your primer and template and bring up to 16ul's | |
| 1 ul of primer + 1 ul of template = 2 ul | |
| 16 - 2 = 14 ul of water needed | |
The Proteomics and Metabolomics Facility will not accept requests for reanalysis of your reaction. Reruns will only be granted if clearly due to Core error.