5'-Thiol on oligos

From: Winnell H. Newman (winnell@ncsu.edu)
Date: Wed Mar 22 2000 - 16:32:51 EST

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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