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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. Reaction mix and 5X buffer can be purchased from the Core Labs.

Protocol For Cycle Sequencing Reaction

Attention: Please contact Mark Dasenko about a new protocol using less Big Dye terminator. The protocol below will work fine, however, there is a new protocol using less Big Dye and will therefore cost less.

  1. In a 96-well PCR plate on ice, put 2 µL of deionized water into each well. If DMSO is needed (2° Structure, high GC, etc.), place 0.5 µL of DMSO and 1.5 µL of water in each well instead.
  2. Now add 1 µL of 5x sequencing buffer to each well.
  3. Next add 5 µL 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.)
  4. Add 2 µL ABI Prism® Big Dye™ Terminator 3.1 reaction mix to each well, making sure not to cross contaminate. (Reaction mix should be thawed on ice prior to use.)
  5. 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.
  6. 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
  7. 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.