To make our life easier, we now aliquot the BigDye 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 BigDye pre-mix (1:12 dilution)
includes either forward or reverse primer, BigDye mix from ABI, DMSO and 5xTM
buffer.
This pre-mix is made by combining:
- 400 ul of forward or reverse primer,
- 300 ul of ABI BigDye (Version 1, #4303151),
- 140 ul of DMSO (Sigma, D8779),
- 380 ul of 5X TM buffer (20 ml 1M Tris-HCl pH9.0 + 0.5 ml 1M MgCl2 + 29.5 ml ddH2O)
- 180 ul dd-water
- 1400 ul final volume
and then dispensing 14 ul into each well of the 96 well colored Robins plates
using the Hydra, and stored frozen at -20degC. After delivering 4 ul of DNA
sequencing template into each well of the 384 well Hydra then is used to transfer
3 ul of this BigDye pre-mix to each sample-containing well in the 384 well viper plate.
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.
- Sign up on the BigDye request form.
- The Green 96 well plates for forward primer.
- The Red 96 well plates for reverse primer.
- 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.
- Using the Hydra, add 4 ul of the 20 ul DNA sequencing template preparation
to each of the 384 wells of the viper plate.
- Place the 96 well Big-Dye pre-mix containing plate in the source position
on Hydra and the 384 well viper containing the already despensed DNA templates
in the other position.
- Run the program, "Transfer Big Dye Mix", and the Hydra will transfer 3ul
BigDye mix to each well. You must make sure the Hydra tips touch the top of
the pre-dispensed DNA sequencing template solution to insure all 3 ul is
transfered. Alternatively, if the Hydra tips do not touch the pre-despensed
DNA sequencing template solution, a manual tip touch can be done by manually
pushing the plate to the side so that the wall of the plate touches the tips
of the Hydra syringes insuring that exactly 3ul of the Big-Dye pre-mix is
transfered to each well of the 384 viper plates.
- 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.
- Please return the colored 96 well Robins plates for re-use.
Bruce Roe, broe@ou.edu