Virtual Reality in SYBR Space
ifao at po.uni-stuttgart.de
Fri Mar 29 08:03:28 EST 1996
Several newsgroup articles on SYBR Green I (see below for references) indicated that the dye
binds tightly to DNA Some articles questioned (or caused me to question) the utility of the dye for preparative
gel applications. I was thus pleased to learn that Gregory Buzard can reamplify DNA from SYBR Green I
stained bands. Are concerns for the use of SYBRed DNA in downstream manipulations invalid (hence the painful
Specifically, does anyone have warnings, experience or tips for:
The reamplification of SYBRed DNA?
Its restriction digestion or cloning (e.g.: might clones from SYBRed DNA have an
increase in mutations over those from EtBr stained DNA?)
The removal of the dye from DNA (see below)?
Its characteristics in DNA-protein binding assays.
Here are some references:
Several article on the subject can be found in the following summary:
From: <a href="mailto:jloring at genpharm.com">jloring at genpharm.com</a> ("Jeanne Loring")<br>
Subject: SYBR Summary<br>
Date: 9 Sep 1994 16:46:18 -0700<br>
Subject: Time:9:19 AM<br>
OFFICE MEMO SYBR Summary Date:9/9/94<br>
(Some excerpts from this summary:) "(Therefore,) I was wondering if the dye somehow complexes
the DNA to the gel matrix?"
"I assume that SBYR green stabilizes the DNA, and remains tightly bound, perhaps changing the<br>
conformation or the charge of the DNA, making it less diffusible. The literature<br>
that comes with the dye says that you can prestain the DNA before running it on<br>
a gel, and that this DOES affect the migration. I assume it affects all DNA<br>
similarly, so putting it in the gel will just alter the migration of both<br>
standards and experimental bands equally, and make no difference. However, I<br>
don't know whether it binds stoichiometrically, or if it EVER comes off, so I've<br>
been loathe to try it for quantitation or prep gels."
Questions to Molecular Probes technical support:
"Me: How do get this (dye) off (my DNA)?<br>
Them: Uh, use standard techniques...<br>
Them: GeneClean should work.<br>
Me: How about n-butanol?<br>
Them: Uh, yeah, that should work too.<br>
Me: Has anybody tried any of this?<br>
Them: Uh....Let me check...I'm not sure.<br>
At this point I thanked them and hung up.
"I guess sunlight is destroying the fluorescent dye, but </i><br>
this should not necessarily be construed as removing the stain from<br>
the DNA. It may well be there but simply no longer be fluorescent,<br>
due to some photochemical change."
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