jloring at genpharm.com
Fri Sep 9 18:46:20 EST 1994
Subject: Time:9:19 AM
OFFICE MEMO SYBR Summary_ Date:9/9/94
Thanks to you who responded to my questions about SYBR Green. It is catalog
#S-7567 from Molecular Probes 4849 Pitchford Ave, Eugene, OR 97402. Phone
503-465-8300, Fax 503 344-6504. It's pricey- at $195 for 1 ml of unknown
concentration in DMSO. It's excited by 254 nm light (your UV lightbox) as well
as 497nm, and emits at 520. We use it routinely now for looking at PCR products
on agarose gels. It is definitely brighter than EtBr, and appears to also
stabilize bands (see discussion below). MP says it is compatible with
restriction enzymes and ligase, but I haven't tried it for a prep gel yet. The
filter sold by Molecular Probes is definitely an advantage when using a polaroid
system. The Wratten filters we use for EtBr (2B and 22) screen out too much of
the signal. However, the orange filter (no identification) that is on our CCD
imaging system (alpha Innotech) works fine. MP's filter isn't anything
special-a cellophane Wratten #15-you can probably find one cheaper in a camera store.
Here is most of the thread on SYBR Green I:
Junhyong (junkim at peaplant.biology.yale.edu (Jun Kim)) wrote:
>Thanks to all on there experience with SYBR Green I. I found that I wasn't
using the proper dilution (didn't let the DMSO melt completely.) Now I get
very nice bright bands and I have gone over completely to using this stain
for routine things (kinda expensive, though). Yesterday, I found an interesting
phenomenon with this dye. I forgot and left my gel staining for >about
15hrs.With EtBr, after this much time in buffer most of the DNA is either gone
ordiffused. Well, I put the gel on the illuminator and found clean nice
>bands!Saved me a lot of trouble, but I was wondering if the dye somehow
complexes theDNA to the gel matrix? I haven't tried putting SYBR Green into the
gel, butthis experience would indicate that it might greatly affect the mobility
of the DNA. Has anybody tried this?
I (Jeanne Loring jloring at genpharm.com) replied:
>YES!! I inadvertently did the same experiment, with a 3% NuSieve TBE gel
containing some <100 bp PCR products. I left it over a weekend in 1:10,000 SYBR
green in water, and on Monday, there was very little diffusion. I assume that
SBYR green stabilizes the DNA, and remains tightly bound, perhaps changing the conformation
or the charge of the DNA, making it less diffusible. The literature that comes
with the dye says that you can prestain the DNA before running it on a gel, and
that this DOES affect the migration. I assume it affects all DNA similarly, so
putting it in the gel will just alter the migration of both standards and
experimental bands equally, and make no difference. However, I don't know
whether it binds stoichiometrically, or if it EVER comes off, so I've been
loathe to try it for quantitation or prep gels. Anyone know?
Junhyong (junkim at peaplant.biology.yale.edu (Jun Kim)) responded:
>I called up Molecular Probes technical support to specifically ask about
this.The conversation went something like,
Me: How do get this off?
Them: Uh, use standard techniques...
Them: GeneClean should work.
Me: How about n-butanol?
Them: Uh, yeah, that should work too.
Me: Has anybody tried any of this?
Them: Uh....Let me check...I'm not sure.
At this point I thanked them and hung up. I guess one of these days, I'll
try band cutting...
Paul N. Hengen[ pnh at ncifcrf.gov | commented on the earlier questions:
>I don't think the DNA is diffusing, but the EtBr is coming off the DNA and
then the bands look fuzzy under UV. I think this because you can re-stain your gel
with EtBr and see good bands again. If you do the same thing with SYBRGreen I,
there is very little diffusion from the DNA and your bands still look"tight"
after several days in buffer. I've noticed though that SYBR Green Idegrades very
quickly when left out at room temperature and goes off within a few days if not
stored in a brown bottle. This makes me think that some wavelength of visible
light can be used to rid the fluorescence (and maybe the DNA binding capacity?)
of the dye. It would be nice if this wavelength were found not to be harmful to
the DNA so that the stain could just be removed by exposure to some specific light
Michael J. Coady (COADY at ERE.UMONTREAL.CA) responded:
>I guess sunlight is destroying the fluorescent dye, but
this should not necessarily be construed as removing the stain from
the DNA. It may well be there but simply no longer be fluorescent,
due to some photochemical change.
And finally, David Hanzel (hanzel at mdyn.com) added this information:
>Just a quick note from the land of Fluorescent Scanners (I work @
Molecular Dynamics) the SYBR dyes from Molecular Probes (Green I & II in
particular) work very well when excited with the 488nm line of an Argon
laser. In many cases they are the BEST dye currently available for staining
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