Sanger Sequencing Services


gel imageThe DNA Core accommodates both single sample users and high-throughput projects. The facility utilizes a 3730xl 96-capillary DNA Analyzer with Applied Biosystems Big Dye Terminator cycle sequencing chemistry. The 96-capillary instrument with robotic plate handler provides a high throughput capacity at minimal cost to investigators. Projects of varying size are accommodated.

Read lengths up to 900 bases per reaction are routine, providing the template is of high quality. Turn around time for submissions is 24 -36 hours. Review DNA Core Policies for a complete description of all core policies relating to services and billing. Contact the DNA Core for additional information.





.........Group........ ......Per Sample Cost......
Single Tube Submission $5.50/reaction
96-well Plate $408 ($4.25/well) *charged as whole plate




Submission Guidelines for Single Samples

  1. gel imageAll samples are to be submitted in a 1.5ml tube. The rack used by our Tecan robotic system restricts us to accepting only tubes with certain dimensions. The DNA Core currently uses Fisher Scientific (Cat. #05408129)1.5ml MCT Graduated tubes for sample submissions. The Fisher Scientific tubes may be purchased from the DNA Core through the Enzyme Freezer Program in a 500 tube per bag quantity.

  2. Total volume of the template + primer mix in the 1.5 ml tube is to be 16 ul.

  3. Recommended total amounts in the 16ul volume for the following DNA templates are:
    • PCR products, 50 - 275 ng (amounts to use when PCR product total length is less than 300 bases otherwise use double stranded DNA amounts listed below)
    • Single-stranded DNA, 275 - 550 ng
    • Double-stranded DNA, 1000 - 2000 ng
    • Large DNA (BAC, YACs, cosmids, and fosmids), 2500 - 4000 ng

  4. Add 15 - 26 pmol of a single primer to each reaction.
  5. *Units Conversion Formula

                   


  6. The 3730 is sensitive to salts and other contamints. Care in template preparation is essential to quality data and instrument maintanence.


Submission Guidelines for 96-well Plate:

  1. Samples are to be submitted in a 96-well plate obtained from the DNA Core. Plates containing less than 96 samples should be loaded in a vertical fashion (A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, etc.)

  2. Total volume of the template + primer mix in each well of the plate is to be 6 ul.

  3. Recommended amounts for the following DNA templates are:
    • PCR products 10 - 50 ng
    • Single-stranded DNA 50 - 100 ng
    • Double-stranded DNA 200 - 500 ng
    • Large DNA (BAC, YACs, cosmids, and fosmids) 500 - 1000 ng

  4. Add 5 - 10 pmol of a single primer to each reaction.

  5. The 3730 is sensitive to salts and other contamints. Care in template preparation is essential to quality data and instrument maintanence.


Guidelines for Properly Labeling Tubes
  1. Sequence orders are placed on-line using the DNA Core's dnaLIMS system. Instructions on the use of the dnaLIMS system, and creation of an account, contact the DNA Core.

  2. When properly submitted each order receives an order number and each sample recieves a requisition number. Label tubes with the last three digits of the requisition number on the top of the tube.
  3. For example: Order #15759 has two sample; Requisition #289573 and Requisition #289574. Each tube is labeled with the last three digits of the requisition #.

    Requisition #289573

    Requisition #289574

  4. Place samples in a plastic bag with the LIMS order number and submitting individual's name clearly written on the outside of the bag. (Plastic bags and permanent marker are provided on a cart outside of room 215).

  5. Place samples in the white refrigeration unit located in the hallway directly across from room 211. A box for samples will be on the left side of the unit on the second shelf from the top.




Primer Guidelines

Primers to be used for automated sequencing must have the correct melting temperature. If the primer/DNA match is 100%, Tm should be 55-60oC (A,T = 2oC each; G,C = 4oC each). Primers, generally, must be 20 bases or longer for use in sequencing reactions. If the match is less complete, Tm must be higher. Avoid primers with long runs of a single base (more than three or four), as well as primers that have secondary structure or can form a dimer. Primer dimer analysis and Tm calculation can be performed using Oligo Analyzer 3.0 on the Integrated DNA Technologies web page. Ensure that your primer concentration is correct and use at least 5 pmol/reaction.



Possible primer problems

  • Primer concentration should be 15-26 pmol in the submitted 1.5ml eppendorf tube. A lower amount of primer can cause a reaction to fail.
  • Poor primer quality will result in poor sequence quality. N-1 species will cause peaks to be out of frame with one another and basecalling software will not be able to assign correct bases.
  • A primer with a melting temperature too low for cycling conditions will not anneal to the template. Design primers with Tm's between 55-60oC for use at the DNA Core.
  • Secondary structure in the primer will cause the primer to self-anneal and not anneal to the template.
  • Mismatch between primer and primer annealing site on the 3' end of a primer can cause the sequencing reaction to fail. If a reaction fails with a standard primer, check to be sure the plasmid contains the primer binding site and that the site was not lost due to restriction digest and cloning of an insert.
  • Multiple priming sites on a template will cause peaks that overlap one another. Basecalling software will not be able to assign a base.



Troubleshooting

The DNA Core staff are always willing to discuss sequencing results. Common issues and sequencing artifacts can be found in a guide prepared by the DNA Core. Our guide, DNA Core Facility: DNA Sequencing Guide, provides illustrations and recommends solutions for several sequencing problems.