DNA Sequencing


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VERY SHORT SURVEY

Our DNA sequencing facility uses an ABI 3130xL Genetic Analyzer to process sequencing samples prepared with ABI’s BigDye® Terminator v3.1 sequencing chemistry. Our 3130xL uses a 50-cm length, 16-capillary array filled with POP-7 high-performance polymer as the separation medium. Our current run method allows for 192 sequencing samples to be processed every 24 hours.

Our lab is located in room C110 in the Microbiology building on the Colorado State University campus. Samples are submitted and data is retrieved using our LIMS website. Please 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 and FAQs .


Premium Level

The premium level is split into two divisions:

1 - The sequencing of large templates that require more Big Dye chemistry than normal sized templates. Larger templates include BAC’s, Cosmids, and Lambda DNA. Please contact Hend Ibrahim regarding genomic sequencing.

  • For BAC, Cosmid, Lambda templates, please provide at least 2 μg of DNA at 0.5 μg/μl.
  • Customer supplied primer for large templates must be at 10 μM or 10 pmol/μl.

2 - Post-PCR clean-up. Our current methods of PCR purification are Qiagen’s Qiaquick PCR purification (column-based) and USB’s Exo-SAP purification (enzyme-based.)

  • Please provide at least 30 μl of sample. Gel electrophoresis must be done prior to submission to verify single-band amplification; the core will perform the PCR clean-up and quantitate the DNA.
  • Customer supplied primers for sequencing should be between 3.2 - 5 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 customer should bring their plasmid or PCR template and primers at the described concentrations. Samples submitted below these concentrations commonly result in reaction failure.

Plasmid template:
Our facility will use 250-300 ng of plasmid per sequencing reaction. If you are submitting a template that will be used for multiple reactions, please provide us with enough template for all reactions.

  • Plasmid DNA under 10 kbp: 0.5 μg per reaction @ 50-100 ng per µl.
  • Plasmid DNA over 10 kbp: 1.0 μg per reaction @ 50-100 ng per µl.

PCR template:
Our facility will use the following amount of PCR product per sequencing reaction:

Under 200 bp - 5 ng
Between 200-500 bp - 15 ng
Between 500-1000 bp - 25 ng
Between 1000-2000 bp - 40 ng
Over 2000 bp - 80-100 ng

If you are submitting a template that will be used in for multiple reactions, please provide us with enough template for all reactions.

  • PCR DNA less than 1000 bases in length; at least 10 µl of DNA @ 10 ng/µl.
  • PCR DNA more than 1000 bases in length; at least 10 µl of DNA @ 20 ng/µl.

Primer:

  • Customer Supplied Primer: 5 μl per reaction @ 3.2 - 5 µM or 3.2 - 5 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 and are comfortable with preparing the sample. 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.

Our facility will add 8 μL of an economy submission. The amount of template and primer listed below is designed to be 2.5x the amount normally used for a sequencing reaction. Our facility asks for the extra amount in case a re-run is needed.

Plasmid template:

  • For Plasmids under 10 kbp: Combine 600 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 10 ng of PCR product per 100 bases and 10 pmol of primer in 20 µl total.

The following is a recommended 4-step 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 5 uM.


Step 1. Determine how much template needs to be added.

DNA needed = (10 ng / 100 bp) * (PCR Size in bp)

DNA needed = (10 ng / 100 bp) * (1000 bp)

DNA needed = 100 ng

Step 2. Determine the amount of μl needed to achieve 100 ng of template.

X μl needed = (DNA needed) / (PCR Concentration)

X μl needed = (100 ng) / (25 ng/μl)

X μl of PCR DNA needed is = 4.0 μl

Step 3. Determine the amount of primer needed.

10 uM = 10 pmol/μl

X μl of primer needed = (10 pmol) / (primer concentration)

X ul = (10 pmol) / (10 pmol/μl)

X = 1 μl

Step 4. Combine your primer and template and bring up to 20 μl.

1 μl of primer + 4 μl of template = 5 μl

20 μl– 5 μl = 15 μl of water needed


NEW! Bulk plates (top)

This service is designed for customers with a large volume of samples. Customers will provide an *approved* 96-well plate 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.

* - The shape of our thermocycler’s and genetic analyzer’s holding trays require a specific-shaped 96-well plate (skirted with a trimmed top-right corner). Our facility recommends ABI’s MicroAmp optical reaction plate, cat# N801-0560, but most similar shaped plates will work.

Plasmid template:

  • For plasmids under 10 kbp: Combine 250 ng of plasmid DNA with 3.2 - 5 pmol of primer in 8 µl total.
  • For plasmids over 10 kbp: Combine 300 ng of plasmid DNA with 3.2 - 5 pmol of primer in 8 µl total.

PCR template:

  • Combine 4 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.

 

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Fort Collins, Colorado 80523
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