Virtual Reality in SYBR Space

[Wayne Coco]

Dear netters,

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
subject heading)?

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.

Thanks,
Wayne

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>
</pre>
<p>
                       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>
Me: Like?<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."