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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)

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 at lwwashin@indiana.edu
(812) 855-8915
Simon Hall 033B

 

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)

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 at lwwashin@indiana.edu
(812) 855-8915
Simon Hall 033B

 

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 product

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.

 

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/

"REACTION & RUN” SEQUENCING

 For the "reaction and run" service, just deliver your template/primer mix for samples you want to sequence and the Sequencing Lab will do the rest. This is how to sign up for this service and deliver samples.

 

Sample sign-up:

Until the web signup is updated, special plate names will be used for “Reaction & Run” plates. On the “Reserve Wells” page  http://dna.bio.indiana.edu/console.php?module=select&action=reserve select a plate labeled in the format seq222222a, seq222222b, etc with an urealistic “Active Date”. Fill out the form as usual.

 

The price is $6.18 per sample.

 

Plate signups will be added as needed. Just look for plates with the seq222222x format. If you have suggestions about the new service, or special requests, please let me know.

 

Sample submission:

1) Primer and Template should be mixed together in 0.2ml strip tubes with strip caps. Tubes must be clearly labeled with the assigned well numbers.

2) Primer amount: 3.2 pico-moles

3) Template amount per below*

4) Bring volume of template/primer mix to 10 uL with 10mM Tris pH 8.0

(These template and primer amounts and volume gives us enough for 2    sequencing reactions)

5) Deliver your template/primer samples to the IMBI by placing sample tubes in the proper wells of the rack labeled "Reactions & Sequencing’ in the IMBI freezer. The rack will have the plate name on it.

 

Template:

*Recommended  Amount

500 bp PCR product

20-40 ng

1 kb PCR product

40-80 ng

2 kb PCR product

80-160 ng

4 kb plasmid

160-320 ng

10 kb plasmid

400-800 ng

  

 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.

Careful measurement of concentrations is critical to getting good results. Inaccurate concentrations are the most common cause of poor reads.

We will run your reactions using these sample set up and cycle sequencing conditions: Half of your supplied template/primer mixes will be used for the sequencing reaction. All reactions will be run using the cycle sequencing program shown below, with  2uL BigDye v3.1. If your DNA template and primer is not suited for this generic program you should do the reactions yourself and submit them as "run only" requests.

 

Temp

Time

96ºC

1 min

96ºC

10 sec

50ºC

5 sec

60ºC

4 min

Cycle 25 times

4ºC

forever

 

Half of your sample will be run and half will be saved for a few days in case a re-run is needed.

Sequencing standards will be loaded on each plate. Failure of the internal standard indicates a poor run and the plate will be re-run at no additional charge.

 



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