Research Facilities:
DNA Sequencing

The DNA sequencer is an ABI 3100 Genetic Analyzer. This new sequencer uses capillary technology instead of the slide gel in the ABI 377 sequencer.

Requirements for DNA samples:

1. Users need to remove all residual salts, proteins, RNAs, and detergents in your DNA samples.
2. Users need to follow the guidelines for template preparation and primer selection.
3. DNA samples should be 100ng/µl concentration and at least 10µl/direction.
4. PRIMERS should be 4µM concentration and at least 15µl. (Note: Users must do their own dilutions of samples and primers)
5. Users need to have correct charge number to process your samples. Without all this information, your DNA sequencing may be delayed.

There are two ways to submit your samples.

1. Submit DNA/primer individually:
• See guidelines for template preparation and primer selection.
  (pdf file)
• Submission form:
  MS Word file | pdf file
2. Premixed DNA with Primer in a tube:
   • See guidelines for premixed primers (pdf file)
   • Submission form:
     MS Word file | pdf file

Turnaround time: Our turnaround time is 3 days from the day of submission, it may increase to 5 days for large number of samples.

Price: $10 / Sequencing
If samples do not work the first time, we will repeat them once without additional charge.

Template and Primer Recommendations for DNA Sequencing

Template preparation guidelines:

1. Concentration and amount of template
   • DS plasmid DNA
     100-300 ng/µl
     1 µg/direction
   • SS or phagemid DNA
     50-100 ng/µl
     500 ng/direction
   • PCR fragment
     10-20 ng/µl for every 200 bases of length. (25-50 for every 500bp PCR length, etc)

   Please give us sufficient DNA/primer for two sequencing reactions. This allows us to repeat a sample in case of technical failure without having to contact you for more samples.

2. For plasmid template preparation, we recommend you to use Qiagen plasmid kits (QiaPrep, Qiagen tip-20, Quagen tip-100, QuaPrep spin, etc.) or the Qiawell plasmid kits. Both give you the best results. In addition to Qiagen procedures, the Ultracentrifugation in CsCl density gradients and the Wizard DNA purification system yield the high quality template DNA.
3. Concentration of template should be based upon OD260 and verified by agarose gel electroresis.
4. Template should be in sterile distilled H2O (preferred) or 10mM tris, pH 8.0, 0.1mM EDTA.

Primer Selection Guidelines:

1. 18-28 nucleotides in length.
2. 50% G/C content.
3. G & C “clamps” on the 3’ and 5’ ends (at least a single G or C residue)
4. Primer should be at least 20-30 bases long at 5’ of region to be sequenced.
5. Avoid multiple Thymidine residues on 3’ and 5’ ends.
6. Avoid primers with long runs (more than 3 or 4) of a single base.
7. Avoid primers with tendency to form strong intramolecular base pairs or primer primer dimers.
8. Melting temperature 55-65°.
9. Check primers for specificity in annealing to template. If possible use a computer program to design primers.
10. PRIMERS SHOULD BE 4µM in CONCENTRATION (For average 20 mer, 4µM corresponds to approximately 27 ng/µl) and at least 15µl.
11. We have following primers in stock: T7, T3, SK, M13-21, M13REV, PCRIIT7, PCRIISP6, SP6, pcDNA3.1 REV, and T7 terminator.

Download a pdf file with these guidelines.

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Guidelines for Premixed DNA primers

Please follow the guidelines to provide template and primer premixed in one tube:

For DS plasmid DNA:

• 600-800 ng ds plasmid DNA template
• 2 µl 4 µM primer
• x µl sterile water
• 28 µl total volume

For PCR Product:

• 10-20 ng templates for every 200 bp of PCR fragment length.
• 2 µl 4uM primer
• x µl sterile water
• 28 µl total volume
  (Mix 25-50 ng for every 500 bp PCR fragment)

This total volume is sufficient for two sequencing reactions, which allow us to repeat a sample in case of technical problems.

Download a pdf file with these guidelines.

For more information, contact Ping Jiang 404-651-0926

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Facilities

Analysis

Computational

Culture

Imaging

Synthesis

Contact information

Ping Jiang 404-651-0926

Downloadable Documents

DNA Preparation Guidelines (pdf)

Submission forms:
Individual DNA/primer
MS Word file | pdf file

Premixed DNA w/ primer
MS Word file | pdf file

Additional Links

Advantaced Biotechnology Core Facilities