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Economy Bulk Sequencing

With economy bulk sequencing, the customer performs the cycle sequencing reaction. The Core Labs cleans and runs the samples on the DNA sequencer. Price depends on how many samples are submitted.  Big Dye Terminator 3.1 reaction mix and 5X buffer can be purchased from the Core Labs.

Protocol For Cycle Sequencing Reaction

 

Attention: Please contact Mark Dasenko if your template is >20kb or very G-rich, as the following protocol may need to be altered.

  1. If you are submitting >48 samples at a time, please use a PCR plate that is compatible with our ABI 3730 sequencer.  The following will work: Cat. No. T-3085-1 from ISC BioExpress or Cat. No. N801-0560 from Applied Biosystems.  In each well of a 96-well PCR plate, put 5 uL of template/primer mixture, using half the amounts of template and primer listed for standard sequencing. (The amount requested for standard sequencing is actually double what is needed.)
  2. Next make a master mix based on the following volumes of reagents per well:
    2 uL deionized water
    1 uL Big Dye Terminator 3.1 (thaw on ice)
    1.5 uL 5X buffer
    0.5 uL DMSO
  3. Now add 5 uL of master mix to each well, making sure not to cross contaminate.
  4. Place a PCR sealing mat or film on the plate and vortex plate slightly at low speed. Centrifuge for a few seconds to get the mixture down to the bottom of each well. We recommend sealing film (Cat.#48461) from Edge BioSystems to prevent sample evaporation. Many other brands do not seal very well or are too sticky to remove.
  5. Using a thermal cycler, heat the plate at 96°C for 5 minutes. Next, subject the plate to 25 cycles under the following conditions:
    96°C for 30 seconds
    50°C for 15 seconds
    60°C for 4 minutes
  6. Bring plate to the Core Labs and place in the refrigerator (not the freezer!). Make sure sample names are clearly marked on the plate. Submit web-order, indicating "Economy Bulk" under sequence type. When filling in the web-order, please submit the samples in the following order: A1, B1, C1 ... H1, A2, B2, etc. Notify Mark Dasenko at least a day in advance before bringing plates to the Core Labs, as samples should be cleaned within 24 hrs of running extension reactions.