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Standard Sequencing

Standard Rate per sample (internal users): $2.18; Reactions with Sequencing: $6.18 (please inquire about volume discount for projects with a minimum of 10,000 samples)

Rate for external users: non-profit is 2X standard rate; for-profit is 3X standard rate

If you wish to use the Institute sequencing facilities, please follow these simple steps.

  1. Sign up for the Sequencer mailing list at
    https://sunflower.bio.indiana.edu/mailman/listinfo/dnasequencer/
    This will let us contact you about matters of importance, such as changes in procedures, additional signup opportunities, etc. You can also communicate directly with other people on the list if you have ideas or questions of general interest.
  2. Purchase an Applied BioSystems BigDye v.3.1 sequencing kit from the Biology Stockroom, or directly from Applied Biosystems.

  3. Follow the recommended protocols below.

  4. Sign up for sequencing at
    http://dna.bio.indiana.edu/
    Username / Password: please contact Lawrence Washington to obtain username and password for the sequencing signup.
  5. Data will be posted the following day at http://dna.bio.indiana.edu/
    If you have any questions at all about sequencing, please contact
    Lawrence Washington
    lwwashin@indiana.edu

    (812) 855-8915
    Simon Hall 033B
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Recommended DNA Sequencing Protocol

Template Prep: DNA templates to be used for sequencing should be free of both organic residues (i.e. phenol and chloroform) and high salts (i.e. DNA precipitated using ethanol and sodium acetate needs to be thoroughly rinsed with 70% ethanol prior to resuspending). Qiagen plasmid prep and PCR clean-up kits produce excellent results provided all rinse steps are followed. It is critical that you know the concentration of your template DNA. Too much template will result in poor reads and possibly damage the capillaries of the ABI3730 machine. We recommend using a spectrophotometer (Nano Drop available for use in the DNA sequencing facility) to accurately measure your DNA concentration.

DNA Sequencing Reactions (final volume=10 µl)

*molarity of template is what counts. Recommended amounts of DNA to use depend on the size of your template. We recommend the following amounts for typical templates. Adjust your amount according to the size of your template:

Template: Recommended Amount
500 bp PCR product 10-20 ng
1 kb PCR product 20-40 ng
2 kb PCR prouct 40-80 ng
4 kb plasmid 80-160 ng
10 kb plasmid 200-400 ng

remember, more is usually not better!

- Perform cycle sequencing according to Big Dye protocol.

- Deliver your samples (the full 10 µL reaction) to the Sequencing Facility in 0.2 mL tubes. If some sample has evaporated, add water to return to 10 µL final volume.

- Reserve wells online at http://dna.bio.indiana.edu/console.php?module=select&action=reserve

- Reservations may be made of edited until 1:00 the day of the run.

- If doing multiple samples, PCR-strip tubes or plates are strongly recommended. Please load samples into strips and plates in the same order/wells as you signed up for on the sequencing web page. This greatly facilitates handling of your samples and reduces chances of sample mix-up.

- Samples must be in the Institute freezer (SI 003) by 3:00 p.m. the day of the run.

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Using CodonCode Aligner to Analyze and Assemble DNA Sequence Data

CodonCode's Aligner program is now available for you to download onto your Mac OS10 or Windows computers. Aligner is a powerful program for assembling DNA sequence contigs from multiple overlapping DNA sequence reads. It can do most everything Sequencher does, and in addition, it can create Phred quality scores for your DNA sequence files (e.g. files generated by the ABI3730 sequencer) and then choose the correct consensus sequence based on the quality scores rather than a simple majority rule. This reduces the amount of manual editing required to generate a "correct" consensus sequence. It also can automatically pick out mutations/polymorphisms in a set of sequences (particularly useful for comparing sequences of PCR-generated clones to a known sequence).

To download Aligner click on the following link and follow the directions: http://aligner.bio.indiana.edu/

To learn how to use Aligner, visit the Aligner quick tour web site: http://www.codoncode.com/aligner/quicktour/

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