How to perform a Big Dye Sequencing?

Описание: Big-Dye Sequencing is a necessary step where dye is added to purified DNA products. These dyes will attach to the nucleotides, therefore the sequencer can detect the sequences by reading these colored nucleotides.

One more step is required before the sequencer can be used. This step will remove the excess dyes left from Big-Dye sequencing. Here's the tutorial on that protocol:    • How to Perform a Pre-Sequencing Bead Clean...  

Protocol to follow:

1) A Master mix should be prepared twice, once with the Forward primer and once with the Reverse primer with the following recipe per sample:

-6.00 µl Sterile H2O
-2.25 µl 5X Big Dye Sequencing Buffer
-0.25 µl Big Dye Terminator V3.1 (This amount can be changed according to the length of sequences. E.g. 0.375 µl is the preferred amount for MediumSeq, and 0.5 µl is the preferred amount for LongSeq.)
-0.50 µl F/R Primer (These primers were used in PCR)

2) Make a Mastermix and add ~9µl of Mastermix solution into each labeled tube.
3) Add 1µl of Post Exo-AP product into labeled tubes.
4) Centrifuge the tubes.
5) Take your samples to the thermocycler and follow the protocol shown in the video.
6) Store your post Big-Dye products at 4 celcius degrees.

For any questions, please leave a comment.