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, Economy, and bulk.
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 Jason Rivest 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: Pick 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 (top)
The standard level is designed to replicate the current level of sequencing service. The customer should bring their plasmid or PCR template and primers at the described concentrations. Template or primers submitted above these concentrations will have to be diluted and will be subject to additional charges. Samples submitted below these concentrations commonly result in reaction failure.
Plasmid template:
- Plasmid DNA under 10 kbp: 0.5 ug per reaction @ 50-100 ng per µl. (Multiple samples should be at the same concentration).
- Plasmid DNA over 10 kbp: 1.0 ug per reaction @ 50-100 ng per µl. (Multiple samples should be at the same concentration)
PCR template:
- PCR DNA less than 1000 bases in length; 10 µl of DNA @ 10 ng/µl.
- PCR DNA more than 1000 bases in length; 10 µl of DNA @ 20 ng/µl.
Primer:
- Customer Supplied Primer: 5 ul per reaction @ 3.2 µM or 3.2 pmol/µl.
Customers may request a re-run of their sample, however, re-runs will be granted at the core's discretion. The amount of DNA described above provides us with enough DNA to perform a re-analysis. Re-runs will only be done if enough sample is provided in the initial submission.
Economy Level (top)
This service is designed for customers who do sequencing regularly. The customer should combine the template and primer at the specified concentrations stated below. Please note core supplied primers are not available for this service.
Plasmid template:
- For Plasmids under 10 kbp: Combine 500 ng of plasmid DNA with 10 pmol of primer in 20 µl total.
- For plasmids over 10 kbp: Combine 750 ng of plasmid DNA with 10 pmol of primer in 20 µl total.
PCR template:
- Combine 5 ng of PCR DNA per 100 bases in length and 10 pmol of primer in 20µl total.
The following is an example of a recommended PCR template setup:
A customer has determined the concentration of their 1000 bp PCR template to be 25 ng/ul. Their primer has a concentration of 10 uM. |
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DNA needed = 5 ng * ( PCR Size / 100 ) |
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DNA needed = 5 ng * ( 1000 / 100 ) |
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DNA needed = 50 ng |
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Second, determine the amount of ul needed to achieve 50 ng of template. |
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X ul needed = (DNA needed) / (PCR Concentration) |
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X ul needed = (50 ng) / (25 ng/ul) |
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X ul of PCR DNA needed is = 2.0 ul |
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Third, determine the amount of primer needed. |
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10 uM = 10 pmol/ul |
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X ul of primer needed = (10 pmol) / (primer concentration) |
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X ul = (10 pmol) / (10 pmol/ul) |
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X = 1 ul |
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Last, combine your primer and template and bring up to 20 ul. |
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1 ul of primer + 2 ul of template = 3 ul |
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20 - 3 = 17 ul of water needed |
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NEW! Bulk plates (top)
This service is designed for customers with a large volume of samples. Customers will provide an approved 96-well plate (the core recommends ABI’s MicroAmp optical reaction plate, cat# N801-0560) containing at least 48 samples with template and primer combined at the specified concentrations stated below. Customers can have up to 96 reactions per plate, but it is recommended at least one well is left open for a control sample to be added for quality control. The core recommends the customer submit one or two economy submissions prior to plate submission to ensure sample quality.
We ask the customer to please email us at least a day before the plate is brought over, so other submitted samples can be affectively organized. Please note core supplied primers are not available for this service.
Plasmid template:
- For plasmids under 10 kbp: Combine 200 ng of plasmid DNA with 3.2 pmol of primer in 8 µl total.
- For plasmids over 10 kbp: Combine 300 ng of plasmid DNA with 3.2 pmol of primer in 8 µl total.
PCR template:
- Combine 2.5 ng of PCR DNA per 100 bases in length and 3.2 pmol of primer in 8 µl total.
Customers may request a re-run of their sample, however, re-runs will be granted at the core's discretion. The amount of DNA described above provides us with enough DNA to perform a re-analysis. Re-runs will only be done if enough sample is provided in the initial submission.