DNA Sequencing Samples

Automated DNA Sequencing Information

An Applied Biosystems Model 3100 automated DNA sequencing unit has been installed in the Protein Chemistry Laboratory, Room 2.226 of the Basic Science Building.   Listed below are some guidelines for successful sample preparation.

1. Contact person: Requests for sequence analysis can be made to Julie Hill , (Ext. 26766) or Esther Surriga, (Ext. 21945)  ISC numbers are now required before sequence analysis can be done.

A. Double Stranded Plasmids: High sample purity and accurate quantitation are absolute requirements for successful automated sequencing. We have had excellent success with plasmids purified using Qiagen mini, midi, maxi, and Qiawell products. Whatever method you choose, it is imperative to completely remove all salt from your samples. This is best achieved by rinsing precipitated plasmids twice with 70% ethanol at room temperature and resuspending samples in ultrapure water. Please don't  use TE buffer!   For each reaction, 1.0 µg of double stranded plasmid in a volume of 10 µl or less is required.

B. Single Stranded Templates: Single-stranded templates typically perform better. Standard methods of template preparation (PEG precipitation followed by repeated phenol extraction) work well. Each reaction requires 0.5 µg in a volume of 5 µl or less.

C. PCR Products: Please contact laboratory staff for optimal conditions and preparation procedures. Roughly 100-500 ng of product is required.

3. Primer considerations: Primer quality is also of critical importance. Standard sequencing primers are in stock and need not be provided by users. Custom designed primers should be chosen using suitable computer software to ensure they are free of hairpin structures and are incapable of self-annealing. Optimal primer length is 19-25 nucleotides. 3.2 pmol of primer in a volume of 2 µl or less are required for each reaction. No special purification is required. Desalted oligos from most suppliers function wonderfully.

4. Sample Submission: If possible, please submit the template DNA and primer pre-mixed in a 0.2ul PCR tube. The total volume should be no more than 12ul.   If you need us to supply one of the standard primers, please have the template in a total volume of 10ul.

5. Charges: The cost of routine sequence analysis is $13.00 per sample. Difficult templates requiring the addition of dGTP or chomosomal DNA will be charged $20. If you are planning a large sequencing project, you may wish to perform the reactions in your own laboratory using your own reagents. These samples can be run at a cost of $10.00 per sample. The facility cannot provide laboratory space or reagents to individuals wishing to perform their own reactions.

6. Turnaround Time: For most templates, this should be 2 working days or less. Users will be contacted when analyses are complete.

7. Acknowledgments: The cost per analysis allows us to recoup supplies and does not include salaries. In effect, the facility is underwriting your research. Because of this, it is important for all of us to document and acknowledge the analyses from this facility. Please submit publications utilizing data from this facility to Dr. Alexander Kurosky.

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