Hi Winnell,
You could try using Ellman's reagent, an aromatic disulfide which reacts
with free thiols to produce a colored by-product that you can measure. Or,
you could use any number of thiol-reactive fluorescent reagents such as
bromomethyl fluorescein to improve your sensitivity. In the latter case, you
would need to separate the unreacted fluorphore from the DNA-fluorphore
product by size exclusion, or organic solvent extraction.
I've used Glen's sulfur-linker products many times, and I've never had any
problems. I suspect your customers may not be using your products optimally.
I have many year's experience in conjugating oligos with surfaces, (which
may be a popular end-use for your products) and most often, the problems
occur at this stage. I am currently working on projects aimed at
immobilizing oligos, and PCR products onto surfaces (planar and beads) for
"genechip" and related applications, and sulfur linkages happen to be an
area I've spent much time on. If you or your customers want more
information, feel free to send an e-mail, I'm glad to help.
Dr. Mark McGovern
"Winnell H. Newman" wrote:
> Hi all:
>
> Any ideas of a quick test I can do as qualitative control to "know" that
> the thiol can be treated and available for use ?
>
> I use Glen Research's great product 5'-Thiol-Modifier C6 S-S (#
> 10-1936-xx) added at 5' end of oligos, do DMTO-ON and poly pak cleanup
> (no TFA treatment).
>
> I run a 20% 8M urea PAGE for QC prior to giving to customer - but
> usually don't do HPLC unless doubts arise.
>
> This works well, but new users sometimes challenge that the oligo is
> proper when the science doesn't work well....
>
> What I mean is this...I usually give the client the oligo with DMTO-ON
> and the S-S disulfide linkage intact. That way the client can prepare a
> portion of the oligo when ready for use, and I like sending it
> "protected" and not treated with DTT to reduce the possibility of
> oxidative disulfide formation.
>
> I will be running HPLC on some recent samples that "appear to be bad" .
> For HPLC I'm going to have 3 samples,
> (1) oligo after poly-pak cleanup (therefore DMTO should be ON)
> (2) oligo treated with TCA or TFA (therefore DMTO should be OFF)
> (3) oligo treated with DTT (therefore thiol will be present)
>
> These should runn differently - right?
>
> But still it won't tell me if the thiol is "useable"....
>
> Any and all suggestions are welcome - even leads to other sources for me
> to do the foot work - please
>
> Thanks
>
> Winnell
>
> --
> Winnell H. Newman, Director, Nucleic Acids Facility (NAF)
> North Carolina State University, Department of Biochemistry
> 128 Polk Hall, Box 7622
> Raleigh, NC 27695-7622
>
> Nucleic Acids Facility Web Page http://www.ncsu.edu/naf
> Email = mailto:winnell@ncsu.edu
> VOX (919) 515-3463
> FAX (919) 515-2047
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