Even-more-diluted BigDye

It turns out that you can use half the amount of BigDye that is recommended by NAPS/SBC for Sanger sequencing and have no noticeable drop in sequence quality. The updated recipe for the working dilutions:

BigDye 3.1 stock      0.5 parts

BigDye buffer            1.5 parts

Water                       1 part

I will prepare all future working dilutions using this recipe and put them in the usual box in the common -20ºC freezer. For more details on how to prepare a sequencing reaction see this post, and for how to purify them see this or this posts.

BigDye 3.1

While cleaning up the freezer a few months back we found encrusted in a block of ice at the bottom of the common lab freezer a box with a seizable amount of BigDye 3.1, the reagent used to prepare samples for Sanger sequencing (basically a PCR mastermix with labelled nucleotides). As that is expensive stuff (what we have is worth 3-4000 dollars), I tested it to see if would still work. All the tests I did were rated “great sequence” when I got the results back from NAPS, so the BigDye is fine.

Thanks to a donation of buffer from NAPS, I have diluted some of the BigDye to the same working concentration NAPS uses (1 part of BigDye, 1.5 parts of buffer, 0.5 parts of water). Follow the instructions on the NAPS website (http://naps.msl.ubc.ca/dna-sequencing/dna-sequencing-services/user-prepared/) to prepare your sample, and use 3 µl of the diluted BigDye. There are eight aliquots of about 150 µl each (50 reactions) in a box with a yellow “BigDye” label in the common -20 ºC freezer. If you plan to do only a few reactions at a time, consider making smaller aliquots for your personal use (BigDye doesn’t like repeated freeze-thaw cycles). To avoid confusion I kept the concentrated BigDye and dilution buffer in their Applied Biosystem box on the door of the first freezer on the right in the freezer room. If we run out of dilution just let me know, and I’ll be happy to prepare more.