384 well DNA sequence reaction pipetting from the 96 well pre-dispensed BigDye (3.0, 3.1) and ET mix plates to 384 well viper plates for BigDye Version 3 and the Amersham ETs.
Shaoping Lin and B. Roe, updated 07-24-06

To make our life easier, we now aliquot both the diluted BigDye Version 3 (or 3.1) and Amersham ET pre-mix into 96 well, Robins plates (Part Number 1047-20-1 for the red 96 well Robins plates and Part Number 1047-20-4 for the green 96 well Robins plates). This pre-mix (1:20 dilution) includes either forward or reverse primer, either BigDye Version 3 mix from ABI or the ET mix from Amersham, DMSO (with BD3.0 only) and 5xTM buffer. With BDv3.1, replace the 50 ul DMSO with 50 ul ddH2O as DMSO seems to have a negative effect on the reaction.

This Amersham ET or BigDye Version 3.0 pre-mix is made by combining:

or, for the BigDye Version 3.1 (ABI # 433692), the pre-mix (1:64 dilution) includes either forward or reverse primer, BigDye Version 3.1 mix from ABI and 5xTM buffer. new 01-24-06

This Big Dye Version 3.1 pre-mix is made by combining:

and then dispensing 10 ul into each well of the 96 well colored Robins plates using the Hydra, and stored frozen at -20degC. The green 96 well Robins plates contain the forward primer BigDye pre-mix. The red 96 well Robins plates contain the reverse primer BigDye pre-mix. Once all the pre-mix has been dispensed from the colored plates, they should be returned so that they can be reused.

  1. Sign up on the BigDye/ET request form.
    1. The Green 96 well plates for forward primer.
    2. The Red 96 well plates for reverse primer.
  2. Remove the 96 well plates from the freezer and let them thaw for a few seconds. Then centrifuge the plates at 1500 rpm for 2 seconds to concentrate the BigDye mix to the bottom of the wells in the 96 well plate.
  3. Place the 96 well Big-Dye V3.0 or ET pre-mix containing plate in the source position on Hydra and the empty 384 well viper on the other position. Note: "Viper" plates are 384 well PCR plates, VWR #47744-810.
  4. Run the program, "Transfer Big Dye v3.0 Mix", and the Hydra will transfer 2 ul BigDye or ET mix to each well.
  5. Then, centrifuge the viper plates at 1500 rpm for 2 seconds to concentrate the reaction mixture at the bottom of the wells in the 384 viper plate.
  6. Using the Hydra, then transfer 4 ul of DNA sequencing template (200 ng) from the Zymark 384-well DNA Sequencing Template isolation protocol that was dissolved in 20 ul of dd water to each of 384 wells of the viper plate containing the already despensed diluted BigDye Version 3.0 or Amersham ET mix.
  7. Centrifuge the viper plates at 1500rpm for 2 seconds to concentrate the reaction mixture at the bottom of the wells in the 384 viper plates.
  8. Immediately transfer the 384 reaction plates to the Viper PCR instrument and initiate the cycle sequencing protocol as recommended by ABI but modified to run for 60 cycles.
  9. Please return the colored 96 well Robins plates for re-use.

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Bruce Roe, broe@ou.edu