dye for DNA

Paul St. Amand PST at KSU.KSU.Edu
Tue Aug 22 10:26:34 EST 1995

In article <DDD03F.5yH at ncifcrf.gov>, pnh at fcs260c2.ncifcrf.gov (Paul N
Hengen) wrote:

PSt> | A company called Molecular Probes Inc. located in Eugene Oregon
sells a green
PSt> | dye which they claim is quite a bit more sensitive than ethidium
bromide for
PSt> | detecting DNA.  I haven't used it, but I am planning to give it a
try within
PSt> | the next couple of months.  So if you try it, let me know how it
well it works
PSt> | for you.
PSt> This topic has been covered here quite extensively. See the FAQ list
for more
PSt> information about SYBR Green I. I've used it alot and really like it,
PSt> there are some shortcomings compared to EtBr. First, if you stain
your gel with
PSt> SYBR Green I (SGI), it is nearly impossible to remove the dye because
you can't
PSt> extract it with an organic solvent like isopropanol or isoamyl
alcohol. I found
PSt> out when I tried to extract DNA from a gel for a mobility shift expt. I was
PSt> forced to use EtBr which I could separate from the DNA for later
shifts. Second,
PSt> it is very expensive and has a very short lifespand. Over a short
time (less
PSt> than one year in DMSO at -20 C) it begins to lose it's effective staining
PSt> ability. I thought at first this might be in part due to freezing and
PSt> but even when I aliquoted it into small volumes of 20 ul and used one at a
PSt> time, the sensitivity dropped and I ended up adding more and more until I
PSt> finally gave up and went back to EtBr to avoid buyng more SGI. Anyone else
PSt> notice this? On the better end, when fresh it is much more sensitive
than EtBr
PSt> and is less mutagenic according to the Ames test.

I have used SYBR Green for about five months and like it. I have not yet
seen problems with it losing its effectiveness. I keep it desiccated at
-20C. I do not stain whole gels however; that is too expensive. I add 2ul
of SYBR Green concentrate to 1600ul of 10X loading buffer (Ficol/BPB/XC)
and store aliquots at -20C. After PCR, I simply quick-thaw the aliquot at
65C, vortex it, add 6 to 8ul to a 50ul PCR reaction, wait 10 min., mix the
sample/dye VERY well, and then run on a gel. It gives very nice bright
bands with no background. Since such a small amount is used per gel, it is
not expensive and even less toxic.

Paul C. St. Amand    (PST at KSU.KSU.Edu)
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