In article <6ri53p$fph at net.bio.net>, PBO <PBO at novo.dk> wrote:
> I recently posted a question concerning removal of residual Big Dye
> terminators and I received many helpful answers. Thank you. All the
> answers are shown below.
Hi,
I have also spent a lot of time testing different methods of precipitation
etc. etc. but I found that the most important factor to avoid the dye
blobs phenomenon was to make sure that you remove _all_ of the ethanol
after precipitation.
When I left my samples to air dry for 10 minutes (and sometimes more) the
DNA would also dry, which made it near impossible to resuspend and thus a
very feint or no signal was detected by our ABI377(XL). sequencer.
Today, my lab either use 7.5M ammonium acetate or 3M sodium acetate (gives
better signal strength, but also increases background peaks from unused
terminators early in the sequence). The most important feature is that
after the ethanol wash supernatant has been aspirated and discraded, we
spin all of the tubes briefly, to collect the ethanol from the sides of
the tube, and aspirate this too. Finally, a couple of minutes in rather
than ten minutes with the lids off will make sure that the ethanol is
removed but your pellet isn't over dried.
Hope this is useful!
Cheers!
Andrew Louka
Gene Technology Group,
Institute of Transplantation Immunology,
The National Hospital University of Oslo,
Norway
