Protein and Nucleic Acid Facility

The facility provides DNA sequencing services using our ABI 3100 16-capillary instrument. We are currently running the POP4 80 cm long read sequencing protocol offering read lengths of up to 800 bp. Data is provided on Zip disk, floppy disk, or via our web server. We can write CDs if necessary, but the CD writer is not on the sequencer's computer so the process is somewhat awkward and time-consuming. Floppy disks can hold data from only four sequences. We will no longer e-mail data because in this age of e-mail viruses and thus e-mail filtering attachments can rarely be sent successfully. In general, samples submitted by 10:00 AM will have a one-day turnaround time. Samples that the customer has performed the sequencing reaction and purification on and which are submitted to us in formamide may be brought to us as late as 3:30 PM. Please contact us in advance if you will bring ready to run samples. Blank DNA sequencing order forms may be obtained from our lab or as a PDF file:
PDF DNA sequencing order form
This is a new form as of 2004-04-05. Please use the current form.

Sequencing from plasmids

To expedite turnaround time and minimize potential errors, templates and primers must be submitted premixed in a clearly labled tube containing 2 µg of plasmid DNA and 13 pmol of primer brought to a total volume of 48 µl with distilled water. If your sample is too dilute to meet these requirements, contact Core Facility staff for instructions. For other types of templates, see below.

Primer Design Guidelines The engineered recombinant Taq polymerase used with the ABI system requires pure primers with a melting temperature greater than 45°C. This enzyme is typically used with an extension temperature of 60°C and an annealing temperature of 50°C so primers used for PCR amplification may not always be optimal for the sequencing reaction.

Primer Design Guidelines

• Primers should be at least 18 bases long to ensure good hybridization.
• Avoid runs of an identical nucleotide, especially runs of four or more Gs.
• Keep the G-C content in the range 30-80%, preferably 50-55%.
• Primers with Tm>45°C produce better results than primers with lower Tm using the Applied Biosystems recommended thermal cycling parameters.
• For primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm>45°C.
• Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA.
• Avoid primers than can hybridize to form dimers.
• Avoid palindromes because they can form secondary structures.
• The primer should be a pure as possible, preferably purified by HPLC.

Sequencing other templates

Mix template with 13 pmol of primer and bring to a total volume of 48 µl with water.

Avoid TE buffer if possible because of the EDTA. Although many samples in TE will work, often failed reactions will work when repeated in the absence of EDTA.

According to Applied Biosystems the following methods of plasmid purification are known to work:

• ABI PRISM plasmid miniprep kit
• Cesium chloride banding
• Modified alkaline lysis/PEG precipitation
• Gentra Systems PureGene kits
• QIAGEN mini, midi, and maxi plasmid kits

It is very important that PCR products to be sequenced be free of primers, dNTPs, enzyme, and buffer, as well as nonspecific PCR products. Nested PCR, semi-nested PCR, or gel purification may be necessary to avoid nonspecific amplification. Recommended methods for purifying PCR products include Centricon-100 columns; QIAquick PCR Purification kits; agarose gel electrophoresis followed by QIAquick Gel Extraction kits; and Shrimp Alkaline Phosphatase and Exonuclease I treatment.

The Applied Biosystems Automated DNA Sequencing Chemistry Guide has more information on template and primer preparation in Chapter 3, pages 39-58. This document is available as a PDF file.

Our standard thermal cycler sequencing program is:

96°C for 1 minute
25 cycles:
96°C for 10 seconds
50°C for 5 seconds
60°C for 4 minutes

This protocol gives good results with most templates using the BigDye kit, which uses a mutated polymerase and dye-labeled dideoxy terminators. If your sample is expected to require a different annealing temperature or other special conditions, please let us know. Be aware that special thermal cycling conditions will impose a delay of at least one extra day because such samples will have to be run separately from the other samples.

Ready to sequence samples

We will also run samples for those users who prefer to do their own sequencing reactions. If you choose to do this please make it clear to us that this is what you are doing when you contact us since very few customers run their own reactions. Contact us if you have any questions.

Data analysis and editing

Free Software for viewing and editing DNA sequencing electropherograms is available.

Fee schedual

Download the sample submission form as a PDF here.

Resequencing policy

Occassionally samples will fail to sequence or will yield truncated sequences shorter than typical. The most frequent reasons for failed or truncated sequences are contaminants in the template and/or primer or incorrect template and/or primer concentration. All sequencing runs contain control template/primer and the first and last samples. If the control samples fail to sequence samples will be resequenced without charge; otherwise, customers are charged for failed samples when controls sequence correctly.